Anti-ctla-4 antibodies and methods of use thereof

ABSTRACT

The instant disclosure provides antibodies that specifically bind to CTLA-4 (e.g., human CTLA-4) and antagonize CTLA-4 function. Also provided are pharmaceutical compositions comprising these antibodies, nucleic acids encoding these antibodies, expression vectors and host cells for making these antibodies, and methods of treating a subject using these antibodies.

1. RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/431,272, filed Dec. 7, 2016, which is incorporated by referenceherein in its entirety.

2. FIELD

The instant disclosure relates to antibodies that specifically bind toCTLA-4 (e.g., human CTLA-4) and methods of using the same.

3. BACKGROUND

T-lymphocytes are central to the adaptive immune response to antigen. Atleast two signals are required for full activation of naive T-cells(Bretscher 1999, Proc Natl Acad Sci USA 96:185-90). A first,antigen-specific signal is provided by interaction of the T-cellreceptor (TCR) with MHC/peptide complex on an antigen-presenting cell(APC). A second, co-stimulatory signal is provided by the interactionsbetween receptors on the T-cell and their ligands on an antigenpresenting cell (APC). Engagement of both TCR/MHC and co-stimulatoryinteractions leads to T-cell activation via a number of intracellularpathways, including calcium-calcineurin and RAS mitogen-activatedprotein kinase, and subsequent activation of transcription factors for anumber of effector compounds, including cytokines such as IL-2. Theseevents lead to T-cell proliferation, generation of a CD4+ helper T-cell(TH) pool, and expansion of activated CD8+ cytotoxic T-cells. Not onlyis co-stimulation critical for full T-cell activation, its absenceduring TCR/MHC engagement results in anergy and/or apoptosis.

Multiple positive and negative co-stimulatory pathways are involved inT-cell regulation; however, the most critical are between CD28 onT-cells and B7-1 (CD80) and B7-2 (CD86) on APCs. CD28 promotes T-celldifferentiation into TH1 phenotype cells and enhances antibodyproduction by B cells and activation of T-cells. B7-1 and B7-2,expressed on APCs such as dendritic cells (DC) and B cells, haveoverlapping but distinct functions. B7-2 is constitutively expressed andis rapidly upregulated on APCs coincident with TCR/MHC engagement(signal 1). B7-1 expression is very low on the resting cell, but istypically induced after prolonged T-cell stimulation. These differencessuggest that while B7-2 may be important in initialization of T-cellactivation, B7-1 may play a greater role in perpetuating the immuneresponse.

After T-cell activation, the negative regulatory receptor CytotoxicT-Lymphocyte Antigen 4 (CTLA-4) is upregulated on T-cells (Alegre etal., 2001, Nat Rev Immunol 1:220-8). CTLA-4 is structurally homologousto CD28 but binds more tightly to both B7-1 and B7-2 ligands. CTLA-4inhibits the immune response in several ways: it competes with CD28 forthe B7 ligands and thus blocks co-stimulation; it negatively signals toinhibit T-cell activation; and it can capture CD80 and CD86 fromopposing cells by trans-endocytosis, resulting in impaired costimulationvia CD28 (Krummel and Allison, 1995, J Exp Med 182:459-465; Walunas etal., 1994, Immunity 1:405-413; Qureshi et al., 2011, Science332:600-603).

Given the critical role of the B7 co-stimulatory pathway in promotingand maintaining an immune response, therapeutic agents designed toantagonize this pathway are promising for the treatment of autoimmunediseases and disorders.

4. SUMMARY

The instant disclosure provides antibodies that specifically bind tohuman CTLA-4 and antagonize CTLA-4 function, e.g., CTLA-4-mediatedimmune suppression. Also provided are pharmaceutical compositionscomprising these antibodies, nucleic acids encoding these antibodies,expression vectors and host cells for making these antibodies, andmethods of treating a subject using these antibodies. The antibodiesdescribed herein are particularly useful for increasing T-cellactivation in response to an antigen (e.g., a tumor antigen or aninfectious disease antigen) and/or decreasing Treg-mediated immunesuppression, and hence for treating cancer in a subject or for treatingor preventing an infectious disease in a subject.

Accordingly, in one aspect the instant disclosure provides an isolatedantibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2, and CDRH3 and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2, and CDRL3, wherein:

(a) CDRH1 comprises the amino acid sequence of SYSMN (SEQ ID NO: 10);(b) CDRH2 comprises the amino acid sequence of SISSSSSYIYYAXSVKG (SEQ IDNO: 18), wherein X is E or D;(c) CDRH3 comprises the amino acid sequence of VGLXGPFDI (SEQ ID NO:19), wherein X is F or M;(d) CDRL1 comprises the amino acid sequence of RASQSVSRYLG (SEQ ID NO:15);(e) CDRL2 comprises the amino acid sequence of GASTRAT (SEQ ID NO: 16);and(f) CDRL3 comprises the amino acid sequence of QQYGSSPWT (SEQ ID NO:17), and wherein the CDRH1, CDRH2, and CDRH3 sequences of the antibodyare not SEQ ID NOs: 10, 11, and 13, respectively.

In certain embodiments, the CDRH2 comprises the amino acid sequence ofSEQ ID NO: 11. In certain embodiments, the CDRH2 comprises the aminoacid sequence of SEQ ID NO: 12. In certain embodiments, the CDRH3comprises the amino acid sequence of SEQ ID NO: 13. In certainembodiments, the CDRH3 comprises the amino acid sequence of SEQ ID NO:14. In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the CDRH1,CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 11,and 14; 10, 12, and 13; or 10, 12, and 14, respectively. In certainembodiments, CDRH1, CDRH2, and CDRH3 comprise the CDRH1, CDRH2, andCDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 12, and 14,respectively. In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 10,11, 14, 15, 16, and 17; 10, 12, 13, 15, 16, and 17; or 10, 12, 14, 15,16, and 17, respectively. In certain embodiments, CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth inSEQ ID NOs: 10, 12, 14, 15, 16, and 17, respectively.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CTLA-4, comprising a heavy chainvariable region comprising complementarity determining regions CDRH1,CDRH2, and CDRH3 and a light chain variable region comprisingcomplementarity determining regions CDRL1, CDRL2, and CDRL3, wherein:

(a) CDRH1 comprises the amino acid sequence of SYSMN (SEQ ID NO: 10);(b) CDRH2 comprises the amino acid sequence of SISSSSSYIYYAXSVKG (SEQ IDNO: 18), wherein X is E or D;(c) CDRH3 comprises the amino acid sequence of VGLXGPFDI (SEQ ID NO:19), wherein X is F or M;(d) CDRL1 comprises the amino acid sequence of RASQSVSRYLG (SEQ ID NO:15);(e) CDRL2 comprises the amino acid sequence of GASTRAT (SEQ ID NO: 16);and(f) CDRL3 comprises the amino acid sequence of QQYGSSPWT (SEQ ID NO:17), and wherein the CDRH1, CDRH2, and CDRH3 sequences of the antibodyare not SEQ ID NOs: 10, 11, and 13, respectively.

In certain embodiments, the CDRH2 comprises the amino acid sequence ofSEQ ID NO: 11. In certain embodiments, the CDRH2 comprises the aminoacid sequence of SEQ ID NO: 12. In certain embodiments, the CDRH3comprises the amino acid sequence of SEQ ID NO: 13. In certainembodiments, the CDRH3 comprises the amino acid sequence of SEQ ID NO:14. In certain embodiments, CDRH1, CDRH2, and CDRH3 comprise the CDRH1,CDRH2, and CDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 11,and 14; 10, 12, and 13; or 10, 12, and 14, respectively. In certainembodiments, CDRH1, CDRH2, and CDRH3 comprise the CDRH1, CDRH2, andCDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 12, and 14,respectively. In certain embodiments, CDRH1, CDRH2, CDRH3, CDRL1, CDRL2,and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 10,11, 14, 15, 16, and 17; 10, 12, 13, 15, 16, and 17; or 10, 12, 14, 15,16, and 17, respectively. In certain embodiments, CDRH1, CDRH2, CDRH3,CDRL1, CDRL2, and CDRL3 comprise the amino acid sequences set forth inSEQ ID NOs: 10, 12, 14, 15, 16, and 17, respectively.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CTLA-4, comprising a heavy chainvariable region comprising complementarity determining regions CDRH1,CDRH2, and CDRH3, and a light chain variable region comprisingcomplementarity determining regions CDRL1, CDRL2, and CDRL3, whereinCDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 comprise the amino acidsequences set forth in SEQ ID NOs: 10, 12, 14, 15, 16, and 17,respectively.

In certain embodiments, the antibody comprises a heavy chain variableregion comprising the amino acid sequence of SEQ ID NO: 20. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising an amino acid sequence which is at least 75%, 80%, 85%, 90%,95%, 99%, or 100% identical to an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2 and 4-8. In certain embodiments, theantibody comprises a heavy chain variable region comprising an aminoacid sequence which is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 3. In certain embodiments, the heavy chain variableregion comprises an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 2 and 4-8. In certain embodiments, the heavychain variable region comprises the amino acid sequence of SEQ ID NO: 8.In certain embodiments, the heavy chain variable region comprises theamino acid sequence of SEQ ID NO: 3. In certain embodiments, theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 23. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 24. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 25. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:26. In certain embodiments, the antibody comprises a heavy chainvariable region having an amino acid sequence derived from a humanIGHV3-21 germline sequence (e.g., IGHV3-21*01, e.g., having amino acidsequence of SEQ ID NO: 21).

In certain embodiments, the antibody comprises a light chain variableregion comprising an amino acid sequence which is at least 75%, 80%,85%, 90%, 95%, 99%, or 100% identical to the amino acid sequence of SEQID NO: 9. In certain embodiments, the antibody comprises a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a light chain comprising theamino acid sequence of SEQ ID NO: 27. In certain embodiments, theantibody comprises a light chain variable region having an amino acidsequence derived from a human IGKV3-20 germline sequence (e.g.,IGKV3-20*01, e.g., having amino acid sequence of SEQ ID NO: 22).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to human CTLA-4, the antibodycomprising a heavy chain variable region having an amino acid sequencederived from a human IGHV3-21 germline sequence (e.g., IGHV3-21*01,e.g., having amino acid sequence of SEQ ID NO: 21), wherein the heavychain variable region comprises the amino acid sequence set forth in SEQID NO: 14. In certain embodiments, the antibody comprises a light chainvariable region having an amino acid sequence derived from a humanIGKV3-20 germline sequence (e.g., IGKV3-20*01, e.g., having amino acidsequence of SEQ ID NO: 22).

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CTLA-4, comprising a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs: 2-8. In certain embodiments, theantibody comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 8. In certain embodiments, the antibodycomprises a heavy chain comprising an amino acid sequence selected fromthe group consisting of SEQ ID NOs: 23-26. In certain embodiments, theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 23. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 24. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 25. In certain embodiments, the antibodycomprises a heavy chain comprising the amino acid sequence of SEQ ID NO:26.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CTLA-4, comprising a heavy chainvariable region and a light chain variable region, wherein the heavychain variable region and the light chain variable region comprise theamino acid sequences set forth in SEQ ID NOs: 2 and 9; 3 and 9; 4 and 9;5 and 9; 6 and 9; 7 and 9; or 8 and 9, respectively. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 8 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 23; and a light chain comprising theamino acid sequence of SEQ ID NO: 27. In certain embodiments, theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 24; and a light chain comprising the amino acid sequence ofSEQ ID NO: 27. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 25; and a lightchain comprising the amino acid sequence of SEQ ID NO: 27. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 26; and a light chain comprising the aminoacid sequence of SEQ ID NO: 27.

In another aspect, the instant disclosure provides an isolated antibodythat specifically binds to human CTLA-4, comprising a heavy chainvariable region having an amino acid sequence derived from a humanIGHV3-21*01 germline sequence (e.g., IGHV3-21*01, e.g., having aminoacid sequence of SEQ ID NO: 21); and a light chain variable regionhaving an amino acid sequence derived from a human IGKV3-20*01 germlinesequence (e.g., IGKV3-20*01, e.g., having amino acid sequence of SEQ IDNO: 22).

In certain embodiments, the antibody comprises a heavy chain constantregion selected from the group consisting of human IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂. In certain embodiments, the heavy chain constantregion is IgG₁. In certain embodiments, the heavy chain constant regionis IgG₂. In certain embodiments, the antibody comprises a light chainconstant region selected from the group consisting of human Igκ and Igλ.

In certain embodiments, the antibody comprises an IgG₁ heavy chainconstant region. In certain embodiments, the antibody comprises a heavychain constant region comprising the amino acid sequence of SEQ ID NO:28. In certain embodiment, the amino acid sequence of the IgG₁ heavychain constant region comprises S239D/I332E mutations, numberedaccording to the EU numbering system. In certain embodiments, theantibody comprises a heavy chain constant region comprising the aminoacid sequence of SEQ ID NO: 29. In certain embodiments, the amino acidsequence of the IgG₁ heavy chain constant region comprisesS239D/A330L/I332E mutations, numbered according to the EU numberingsystem. In certain embodiments, the antibody comprises a heavy chainconstant region comprising the amino acid sequence of SEQ ID NO: 30. Incertain embodiments, the amino acid sequence of the IgG₁ heavy chainconstant region comprises L235V/F243L/R292P/Y300L/P396L mutations,numbered according to the EU numbering system. In certain embodiments,the antibody comprises a heavy chain constant region comprising theamino acid sequence of SEQ ID NO: 31. In certain embodiments, the IgG₁heavy chain constant region is afucosylated IgG₁.

In certain embodiments, the antibody comprises a human IgG heavy chainconstant region that is a variant of a wild type human IgG heavy chainconstant region, wherein the variant human IgG heavy chain constantregion binds to FcγRIIIA with a higher affinity than the wild type humanIgG heavy chain constant region binds to FcγRIIIA. In certainembodiments, the variant human IgG heavy chain constant region is avariant human IgG₁ heavy chain constant region.

In certain embodiments, the antibody comprises a light chain constantregion selected from the group consisting of human Igκ and Igλ. Incertain embodiments, the antibody comprises an Igκ light chain constantregion. In certain embodiments, the antibody comprises a light chainconstant region comprising the amino acid sequence of SEQ ID NO: 32.

In another aspect, the instant disclosure provides an isolated antibodythat cross-competes for binding to human CTLA-4 with an antibodydescribed herein. In another aspect, the instant disclosure provides anisolated antibody that cross-competes for binding to human CTLA-4 withan antibody comprising the heavy and light chain variable region aminoacid sequences set forth in SEQ ID NOs: 8 and 9, respectively.

In another aspect, the instant disclosure provides an isolated antibodythat binds to the same epitope on human CTLA-4 as an antibody describedherein. In another aspect, the instant disclosure provides an isolatedantibody that binds to the same epitope on human CTLA-4 as an antibodycomprising the heavy and light chain variable region amino acidsequences set forth in SEQ ID NOs: 8 and 9, respectively.

In another aspect, the instant disclosure provides an isolated antibodythat binds, e.g., specifically binds, to an epitope of human CTLA-4. Incertain embodiments, the antibody binds to an epitope located within aregion of human CTLA-4 consisting of an amino acid sequence selectedfrom the group consisting of SEQ ID NOs: 34-39. In certain embodiments,the antibody binds to an epitope located within a region of human CTLA-4consisting of the amino acid sequence of SEQ ID NO: 37. In certainembodiments, the antibody binds to an epitope located within a region ofhuman CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 36. Incertain embodiments, the antibody binds to an epitope located within aregion of human CTLA-4 consisting of the amino acid sequence of SEQ IDNO: 35. In certain embodiments, the antibody binds to an epitope locatedwithin a region of human CTLA-4 consisting of the amino acid sequence ofSEQ ID NO: 34. In certain embodiments, the antibody binds to an epitopelocated within a region of human CTLA-4 consisting of the amino acidsequence of SEQ ID NO: 38. In certain embodiments, the antibody binds toan epitope located within a region of human CTLA-4 consisting of theamino acid sequence of SEQ ID NO: 39.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanCTLA-4 as any antibody of the present invention. In certain embodiments,the antibody binds to an epitope located within a region of human CTLA-4consisting of an amino acid sequence selected from the group consistingof SEQ ID NOs: 34-39. In certain embodiments, the antibody binds to anepitope located within a region of human CTLA-4 consisting of the aminoacid sequence of SEQ ID NO: 37. In certain embodiments, the antibodybinds to an epitope located within a region of human CTLA-4 consistingof the amino acid sequence of SEQ ID NO: 36. In certain embodiments, theantibody binds to an epitope located within a region of human CTLA-4consisting of the amino acid sequence of SEQ ID NO: 35. In certainembodiments, the antibody binds to an epitope located within a region ofhuman CTLA-4 consisting of the amino acid sequence of SEQ ID NO: 34. Incertain embodiments, the antibody binds to an epitope located within aregion of human CTLA-4 consisting of the amino acid sequence of SEQ IDNO: 38. In certain embodiments, the antibody binds to an epitope locatedwithin a region of human CTLA-4 consisting of the amino acid sequence ofSEQ ID NO: 39.

In another aspect, the instant disclosure provides an antibody that,when bound to a human CTLA-4 protein or fragment thereof, e.g.,comprising the amino acid sequence of residues 37-162 of SEQ ID NO: 33,reduces hydrogen/deuterium exchange in a region consisting of the aminoacid sequence set forth in SEQ ID NO: 34 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 34 in the absence of the antibody, as determined by ahydrogen/deuterium assay. In another aspect, the instant disclosureprovides an antibody that, when bound to a human CTLA-4 protein orfragment thereof, e.g., comprising the amino acid sequence of residues37-162 of SEQ ID NO: 33, reduces hydrogen/deuterium exchange in a regionconsisting of the amino acid sequence set forth in SEQ ID NO: 35relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 35 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human CTLA-4 protein or fragment thereof, e.g., comprising the aminoacid sequence of residues 37-162 of SEQ ID NO: 33, reduceshydrogen/deuterium exchange in a region consisting of the amino acidsequence set forth in SEQ ID NO: 36 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 36 in the absence of the antibody, as determined by ahydrogen/deuterium assay. In another aspect, the instant disclosureprovides an antibody that, when bound to a human CTLA-4 protein orfragment thereof, e.g., comprising the amino acid sequence of residues37-162 of SEQ ID NO: 33, reduces hydrogen/deuterium exchange in a regionconsisting of the amino acid sequence set forth in SEQ ID NO: 37relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 37 in the absence of theantibody, as determined by a hydrogen/deuterium assay. In anotheraspect, the instant disclosure provides an antibody that, when bound toa human CTLA-4 protein or fragment thereof, e.g., comprising the aminoacid sequence of residues 37-162 of SEQ ID NO: 33, reduceshydrogen/deuterium exchange in a region consisting of the amino acidsequence set forth in SEQ ID NO: 38 relative to hydrogen/deuteriumexchange in the region consisting of the amino acid sequence set forthin SEQ ID NO: 38 in the absence of the antibody, as determined by ahydrogen/deuterium assay. In another aspect, the instant disclosureprovides an antibody that, when bound to a human CTLA-4 protein orfragment thereof, e.g., comprising the amino acid sequence of residues37-162 of SEQ ID NO: 33, reduces hydrogen/deuterium exchange in a regionconsisting of the amino acid sequence set forth in SEQ ID NO: 39relative to hydrogen/deuterium exchange in the region consisting of theamino acid sequence set forth in SEQ ID NO: 39 in the absence of theantibody, as determined by a hydrogen/deuterium assay.

In another aspect, the instant disclosure provides an antibody orisolated antibody that specifically binds to the same epitope of humanCTLA-4 as any antibody of the present invention. In certain embodiments,the antibody, when bound to a human CTLA-4 protein or fragment thereof,e.g., comprising the amino acid sequence of residues 37-162 of SEQ IDNO: 33, reduces hydrogen/deuterium exchange in a region consisting ofthe amino acid sequence set forth in SEQ ID NO: 34 relative tohydrogen/deuterium exchange in the region consisting of the amino acidsequence set forth in SEQ ID NO: 34 in the absence of the antibody, asdetermined by a hydrogen/deuterium assay. In certain embodiments, theantibody, when bound to a human CTLA-4 protein or fragment thereof,e.g., comprising the amino acid sequence of residues 37-162 of SEQ IDNO: 33, reduces hydrogen/deuterium exchange in a region consisting ofthe amino acid sequence set forth in SEQ ID NO: 35 relative tohydrogen/deuterium exchange in the region consisting of the amino acidsequence set forth in SEQ ID NO: 35 in the absence of the antibody, asdetermined by a hydrogen/deuterium assay. In certain embodiments, theantibody, when bound to a human CTLA-4 protein or fragment thereof,e.g., comprising the amino acid sequence of residues 37-162 of SEQ IDNO: 33, reduces hydrogen/deuterium exchange in a region consisting ofthe amino acid sequence set forth in SEQ ID NO: 36 relative tohydrogen/deuterium exchange in the region consisting of the amino acidsequence set forth in SEQ ID NO: 36 in the absence of the antibody, asdetermined by a hydrogen/deuterium assay. In certain embodiments, theantibody, when bound to a human CTLA-4 protein or fragment thereof,e.g., comprising the amino acid sequence of residues 37-162 of SEQ IDNO: 33, reduces hydrogen/deuterium exchange in a region consisting ofthe amino acid sequence set forth in SEQ ID NO: 37 relative tohydrogen/deuterium exchange in the region consisting of the amino acidsequence set forth in SEQ ID NO: 37 in the absence of the antibody, asdetermined by a hydrogen/deuterium assay. In certain embodiments, theantibody, when bound to a human CTLA-4 protein or fragment thereof,e.g., comprising the amino acid sequence of residues 37-162 of SEQ IDNO: 33, reduces hydrogen/deuterium exchange in a region consisting ofthe amino acid sequence set forth in SEQ ID NO: 38 relative tohydrogen/deuterium exchange in the region consisting of the amino acidsequence set forth in SEQ ID NO: 38 in the absence of the antibody, asdetermined by a hydrogen/deuterium assay. In certain embodiments, theantibody, when bound to a human CTLA-4 protein or fragment thereof,e.g., comprising the amino acid sequence of residues 37-162 of SEQ IDNO: 33, reduces hydrogen/deuterium exchange in a region consisting ofthe amino acid sequence set forth in SEQ ID NO: 39 relative tohydrogen/deuterium exchange in the region consisting of the amino acidsequence set forth in SEQ ID NO: 39 in the absence of the antibody, asdetermined by a hydrogen/deuterium assay.

In certain embodiments, the antibody is a human antibody. In certainembodiment, the antibody is a bispecific antibody.

In certain embodiments, the antibody is antagonistic to human CTLA-4. Incertain embodiments, the antibody deactivates, reduces, or inhibits anactivity of human CTLA-4. In certain embodiments, the antibody inhibitsbinding of human CTLA-4 to human CD80 or human CD86. In certainembodiments, the antibody induces IL-2 production by peripheral bloodmononuclear cells (PBMCs) stimulated with staphylococcal enterotoxin A(SEA).

In certain embodiments, the antibody is conjugated to a cytotoxic agent,cytostatic agent, toxin, radionuclide, or detectable label.

In certain embodiments, the N-terminal amino acid residue of the heavychain variable region and/or the light chain variable region of theantibody has been converted to pyroglutamate.

In one embodiment, the present invention relates to an antibody of thepresent invention for use as a medicament.

In one embodiment, the present invention relates to use of an antibodyof the present invention for preparing pharmaceutical compositions ormedicaments for immunotherapy. In certain embodiments, the immunotherapyis for increasing T-cell activation in response to an antigen in asubject, optionally for treating cancer, or treating or preventinginfectious diseases.

In one embodiment, the present invention relates to an antibody of thepresent invention for use as a diagnostic.

In one embodiment, the present invention relates to the use of anantibody of the present invention for in vitro detection of human CTLA-4in a biological sample.

In another aspect, the instant disclosure provides a pharmaceuticalcomposition comprising an anti-CTLA-4 antibody described herein and apharmaceutically acceptable carrier or excipient.

In another aspect, the instant disclosure provides an isolatedpolynucleotide encoding a heavy and/or light chain of an antibodydescribed herein. In another aspect, the instant disclosure provides avector comprising the polynucleotide. In another aspect, the instantdisclosure provides a recombinant host cell comprising thepolynucleotide or the vector. In another aspect, the instant disclosureprovides a method of producing an antibody that binds to human CTLA-4,the method comprising culturing the host cell so that the polynucleotideis expressed and the antibody is produced.

In another aspect, the instant disclosure provides a method ofincreasing T-cell activation in response to an antigen in a subject, themethod comprising administering to the subject an effective amount of ananti-CTLA-4 antibody or pharmaceutical composition described herein. Inanother aspect, the instant disclosure provides a method of treatingcancer in a subject, the method comprising administering to the subjectan effective amount of an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein.

In certain embodiments, the subject has cancer. In certain embodiments,the subject has a metastatic or locally advanced tumor (e.g., solidtumor). In certain embodiments, the cancer is treated in accordance witha method described herein as a first cancer therapy after diagnosis ofthe metastatic or locally advanced tumor (e.g., within 1, 2, 3, 4, 5, or6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12months after diagnosis). In certain embodiments, the cancer is treatedin accordance with a method described herein as the first cancer therapyafter diagnosis of tumor progression (e.g., within 1, 2, 3, 4, 5, or 6days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 monthsafter diagnosis of tumor progression) that has occurred despite previoustreatment of the tumor with a different cancer therapy, optionallywherein the method described herein is provided as the second cancertherapy administered. In certain embodiments, the cancer is treated inaccordance with a method described herein as the first cancer therapyafter diagnosis of toxicity of a different cancer therapy (e.g., within1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4,6, 8, or 12 months after diagnosis of toxicity of the different cancertherapy), optionally wherein the method described herein is provided asthe second cancer therapy administered. In certain embodiments, thecancer treated in accordance with the methods described herein is ametastatic or locally advanced cancer (e.g., solid tumor) for which nostandard therapy is available. In other embodiments, the cancer treatedin accordance with the methods described herein is a metastatic orlocally advanced cancer (e.g., solid tumor) for which a standard therapyhas failed (i.e., the cancer has progressed after the standard therapy).In certain embodiments, a therapy fails if the cancer is refractory tothe therapy. In certain embodiments, a therapy fails if the cancerrelapses after responding, fully or partially, to the therapy. Incertain embodiments, metastatic or locally advanced cancer (e.g., solidtumor) has been confirmed histologically or cytologically.

In certain embodiments, the cancer expresses PD-L1. In certainembodiments, the percentage of tumor cells in a sample of the cancerthat exhibit detectable membrane expression (e.g., partial or completemembrane expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%).In certain embodiments, the percentage of tumor cells in a sample of thecancer that exhibit detectable membrane expression (e.g., partial orcomplete membrane expression) of PD-L1 is at least 1%. In certainembodiments, the percentage of tumor cells in a sample of the cancerthat exhibit detectable membrane expression (e.g., partial or completemembrane expression) of PD-L1 is at least 5%. In certain embodiments,the percentage of tumor cells in a sample of the cancer that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 25%. In certain embodiments, thepercentage of tumor cells in a sample of the cancer that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 50%.

In certain embodiments, the metastatic or locally advanced tumorexpresses PD-L1. In certain embodiments, the percentage of tumor cellsin a sample of the metastatic or locally advanced tumor that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%). Incertain embodiments, the percentage of tumor cells in a sample of themetastatic or locally advanced tumor that exhibit detectable membraneexpression (e.g., partial or complete membrane expression) of PD-L1 isat least 1%. In certain embodiments, the percentage of tumor cells in asample of the metastatic or locally advanced tumor that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 5%. In certain embodiments, thepercentage of tumor cells in a sample of the metastatic or locallyadvanced tumor that exhibit detectable membrane expression (e.g.,partial or complete membrane expression) of PD-L1 is at least 25%. Incertain embodiments, the percentage of tumor cells in a sample of themetastatic or locally advanced tumor that exhibit detectable membraneexpression (e.g., partial or complete membrane expression) of PD-L1 isat least 50%.

In certain embodiments, the cancer is a cervical cancer. In certainembodiments, the cancer is a metastatic or locally advanced cancer(e.g., solid tumor). In certain embodiments, the metastatic or locallyadvanced cancer (e.g., solid tumor) is a metastatic or locally advanced,unresectable squamous cell carcinoma, adenosquamous carcinoma, oradenocarcinoma of the cervix. In certain embodiments, no standardtherapy is available for the cancer (e.g., cervical cancer) ormetastatic or locally advanced tumor (e.g., solid tumor). In certainembodiments, the cancer (e.g., cervical cancer) or metastatic or locallyadvanced tumor (e.g., solid tumor) is refractory to a standard therapy.In certain embodiments, the cancer (e.g., cervical cancer) or metastaticor locally advanced tumor (e.g., solid tumor) has relapsed after astandard therapy. In certain embodiments, the standard therapy comprisesa platinum-containing chemotherapy. In certain embodiments, the standardtherapy is a platinum-containing doublet. In certain embodiments, thecancer (e.g., cervical cancer) is a metastatic or locally advanced,unresectable squamous cell carcinoma, adenosquamous carcinoma, oradenocarcinoma of the cervix that has relapsed after aplatinum-containing doublet administered for treatment of advanced(recurrent, unresectable, or metastatic) disease. In certainembodiments, the cancer (e.g., cervical cancer) or metastatic or locallyadvanced tumor is HPV positive. In certain embodiments, the cancer ormetastatic or locally advanced solid tumor is head and neck cancer,melanoma, renal cell carcinoma, urothelial carcinoma, or endometrialcarcinoma. In certain embodiments, the cancer (e.g., cervical cancer) ormetastatic or locally advanced solid tumor is associated withmicrosatellite instability.

In certain embodiments, the subject has cervical cancer (e.g., ametastatic or locally advanced, unresectable squamous cell carcinoma,adenosquamous carcinoma, or adenocarcinoma of the cervix), and themethod comprises administering to the subject an effective amount of ananti-CTLA-4 antibody described herein, e.g., AGEN1884.H3 (IgG₁S239D/A330L/I332E), or pharmaceutical composition comprising suchanti-CTLA-4 antibody as a first cancer therapy after diagnosis of thecervical cancer (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6,8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis),optionally wherein the anti-CTLA-4 antibody described herein, e.g.,AGEN1884.H3 (IgG₁ S239D/A330L/I332E), or pharmaceutical compositioncomprising such anti-CTLA-4 antibody is administered at the dosage andfrequency selected from the group consisting of 0.3 mg/kg every fourweeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks, 0.3 mg/kgevery six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3mg/kg every twelve weeks, 1 mg/kg every twelve weeks, and 3 mg/kg everytwelve weeks. In certain embodiments, the subject has cervical cancer(e.g., a metastatic or locally advanced, unresectable squamous cellcarcinoma, adenosquamous carcinoma, or adenocarcinoma of the cervix),and the method comprises administering to the subject an effectiveamount of a therapeutic combination comprising an anti-CTLA-4 antibodydescribed herein, e.g., AGEN1884.H3 (IgG₁ S239D/A330L/I332E), orpharmaceutical composition comprising such anti-CTLA-4 antibody, andpembrolizumab as a first cancer therapy after diagnosis of the cervicalcancer (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis), optionallywherein the anti-CTLA-antibody described herein, e.g., AGEN1884.H3 (IgG₁S239D/A330L/I332E), or pharmaceutical composition comprising suchanti-CTLA-4 antibody, is administered at the dosage and frequencyselected from the group consisting of 0.3 mg/kg every four weeks, 1mg/kg every four weeks, 3 mg/kg every four weeks, 0.3 mg/kg every sixweeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3 mg/kg everytwelve weeks, 1 mg/kg every twelve weeks, and 3 mg/kg every twelveweeks, and pembrolizumab is administered at 200 mg every three weeks.

In certain embodiments, the cancer is a non-small cell lung cancer(NSCLC). In certain embodiments, the NSCLC is a Stage IV NSCLC. Incertain embodiments, the percentage of tumor cells in a sample of theNSCLC that exhibit detectable membrane expression (e.g., partial orcomplete membrane expression) of PD-L1 is at least 1% (e.g., at least2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%,80%, or 90%). In certain embodiments, the percentage of tumor cells in asample of the NSCLC that exhibit detectable membrane expression (e.g.,partial or complete membrane expression) of PD-L1 is at least 1%. Incertain embodiments, the percentage of tumor cells in a sample of theNSCLC that exhibit detectable membrane expression (e.g., partial orcomplete membrane expression) of PD-L1 is at least 5%. In certainembodiments, the percentage of tumor cells in a sample of the NSCLC thatexhibit detectable membrane expression (e.g., partial or completemembrane expression) of PD-L1 is at least 25%. In certain embodiments,the percentage of tumor cells in a sample of the NSCLC that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 50%. In certain embodiments, the NSCLChas no EGFR or ALK genomic tumor aberrations. In certain embodiments,the metastatic or locally advanced NSCLC has no EGFR sensitizingmutation (e.g., mutation that is amenable to treatment with a tyrosinekinase inhibitor including erlotinib, gefitinib, or afatanib) or ALKtranslocation. In certain embodiments, the subject has received no priorsystemic chemotherapy treatment for NSCLC.

In certain embodiments, the metastatic or locally advanced solid tumoris a metastatic or locally advanced non-small cell lung cancer (NSCLC).In certain embodiments, the metastatic or locally advanced solid tumoris a metastatic non-small cell lung cancer (NSCLC). In certainembodiments, the metastatic or locally advanced solid tumor is a StageIV, metastatic or locally advanced NSCLC. In certain embodiments, themetastatic or locally advanced solid tumor is a Stage IV, metastaticNSCLC. In certain embodiments, the percentage of tumor cells in a sampleof the metastatic or locally advanced NSCLC that exhibit detectablemembrane expression (e.g., partial or complete membrane expression) ofPD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%). In certain embodiments,the percentage of tumor cells in a sample of the metastatic or locallyadvanced NSCLC that exhibit detectable membrane expression (e.g.,partial or complete membrane expression) of PD-L1 is at least 1%. Incertain embodiments, the percentage of tumor cells in a sample of themetastatic or locally advanced NSCLC that exhibit detectable membraneexpression (e.g., partial or complete membrane expression) of PD-L1 isat least 5%. In certain embodiments, the percentage of tumor cells in asample of the metastatic or locally advanced NSCLC that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 25%. In certain embodiments, thepercentage of tumor cells in a sample of the metastatic or locallyadvanced NSCLC that exhibit detectable membrane expression (e.g.,partial or complete membrane expression) of PD-L1 is at least 50%. Incertain embodiments, the metastatic or locally advanced NSCLC has noEGFR or ALK genomic tumor aberrations. In certain embodiments, thesubject has received no prior systemic chemotherapy treatment formetastatic or locally advanced NSCLC.

In certain embodiments, the subject has NSCLC (e.g., Stage IV,metastatic, or locally advanced NSCLC), optionally wherein thepercentage of tumor cells in a sample of the NSCLC that exhibitdetectable expression (e.g., membrane expression, partial or completemembrane expression) of PD-L1 is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%, and the methodcomprises administering to the subject an effective amount of ananti-CTLA-4 antibody described herein, e.g., AGEN1884.H3 (IgG₁S239D/A330L/I332E), or pharmaceutical composition comprising suchanti-CTLA-4 antibody, as a first cancer therapy after diagnosis of thecervical cancer (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6,8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis),optionally wherein the anti-CTLA-4 antibody described herein, e.g.,AGEN1884.H3 (IgG₁ S239D/A330L/I332E), or pharmaceutical compositioncomprising such anti-CTLA-4 antibody, is administered at the dosage andfrequency selected from the group consisting of 0.3 mg/kg every fourweeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks, 0.3 mg/kgevery six weeks, 1 mg/kg every six weeks, 3 mg/kg every six weeks, 0.3mg/kg every twelve weeks, 1 mg/kg every twelve weeks, and 3 mg/kg everytwelve weeks. In certain embodiments, the subject has NSCLC (e.g., StageIV, metastatic, or locally advanced NSCLC), optionally wherein thepercentage of tumor cells in a sample of the NSCLC that exhibitdetectable expression (e.g., membrane expression, partial or completemembrane expression) of PD-L1 is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%, and the methodcomprises administering to the subject a therapeutic combinationcomprising an anti-CTLA-4 antibody described herein, e.g., AGEN1884.H3(IgG₁ S239D/A330L/I332E), or pharmaceutical composition comprising suchanti-CTLA-4 antibody, and pembrolizumab as a first cancer therapy afterdiagnosis of the cervical cancer (e.g., within 1, 2, 3, 4, 5, or 6 days;1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months afterdiagnosis), optionally wherein the anti-CTLA-antibody described herein,e.g., AGEN1884.H3 (IgG₁ S239D/A330L/I332E), or pharmaceuticalcomposition comprising such anti-CTLA-4 antibody, is administered at thedosage and frequency selected from the group consisting of 0.3 mg/kgevery four weeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks,0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every sixweeks, 0.3 mg/kg every twelve weeks, 1 mg/kg every twelve weeks, and 3mg/kg every twelve weeks, and pembrolizumab is administered at 200 mgevery three weeks.

In certain embodiments, the cancer is a cutaneous squamous-cellcarcinoma (cSCC). In certain embodiments, the metastatic or locallyadvanced solid tumor is a Stage IV cutaneous squamous-cell carcinoma(cSCC). In certain embodiments, the cSCC is diagnosed histologically orcytologically according to the eighth edition of the American JointCommittee on Cancer staging manual. In certain embodiments, the cSCC isnot curable with radiation therapy. In certain embodiments, the subjecthas cSCC (e.g., Stage IV cSCC), and the method comprises administeringto the subject an effective amount of an anti-CTLA-4 antibody describedherein, e.g., AGEN1884.H3 (IgG₁ S239D/A330L/I332E), or pharmaceuticalcomposition comprising such anti-CTLA-4 antibody, as a first cancertherapy after diagnosis of the cSCC (e.g., within 1, 2, 3, 4, 5, or 6days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 monthsafter diagnosis), optionally wherein the anti-CTLA-4 antibody describedherein, e.g., AGEN1884.H3 (IgG₁ S239D/A330L/I332E), or pharmaceuticalcomposition comprising such anti-CTLA-4 antibody, is administered at thedosage and frequency selected from the group consisting of 0.3 mg/kgevery four weeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks,0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every sixweeks, 0.3 mg/kg every twelve weeks, 1 mg/kg every twelve weeks, and 3mg/kg every twelve weeks. In certain embodiments, the subject has cSCC(e.g., Stage IV cSCC), and the method comprises administering to thesubject an effective amount of a therapeutic combination comprising ananti-CTLA-4 antibody described herein, e.g., AGEN1884.H3 (IgG₁S239D/A330L/I332E), or pharmaceutical composition comprising suchanti-CTLA-4 antibody, and pembrolizumab as a first cancer therapy afterdiagnosis of the cSCC (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3,4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months afterdiagnosis), optionally wherein the anti-CTLA-4 antibody describedherein, e.g., AGEN1884.H3 (IgG₁ S239D/A330L/I332E), or pharmaceuticalcomposition comprising such anti-CTLA-4 antibody, is administered at thedosage and frequency selected from the group consisting of 0.3 mg/kgevery four weeks, 1 mg/kg every four weeks, 3 mg/kg every four weeks,0.3 mg/kg every six weeks, 1 mg/kg every six weeks, 3 mg/kg every sixweeks, 0.3 mg/kg every twelve weeks, 1 mg/kg every twelve weeks, and 3mg/kg every twelve weeks, and pembrolizumab is administered at 200 mgevery three weeks.

In certain embodiments, the anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered intravenously. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered intravenously at 0.01 mg/kg, 0.03mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg,optionally at an interval of once every two weeks. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered intravenously at 0.01 mg/kg, 0.03mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg,optionally at an interval of once every three weeks. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered intravenously at 0.01 mg/kg, 0.03mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg,optionally at an interval of once every four weeks. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered intravenously at 0.01 mg/kg, 0.03mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg,optionally at an interval of once every six weeks. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered intravenously at 0.01 mg/kg, 0.03mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or 10 mg/kg,optionally at an interval of once every twelve weeks. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered intratumorally. In certain embodiments,the anti-CTLA-4 antibody or pharmaceutical composition described hereinis administered intratumorally at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3mg/kg, 1 mg/kg, or 3 mg/kg, optionally at an interval of once everythree weeks. In certain embodiments, the anti-CTLA-4 antibody orpharmaceutical composition described herein is administeredintratumorally at 0.03 mg/kg, 0.1 mg/kg, or 0.3 mg/kg, optionally at aninterval of once every three weeks. In certain embodiments, theanti-CTLA-4 antibody or pharmaceutical composition described herein isadministered intratumorally at a dose that is up to 5-fold, 10-fold,20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold,100-fold, or 200-fold lower than a dose given by systemicadministration. In certain embodiments, the anti-CTLA-4 antibody orpharmaceutical composition described herein is administeredintratumorally at a dose that is up to 10-fold lower than a dose givenby systemic administration. In certain embodiments, the anti-CTLA-4antibody or pharmaceutical composition described herein is administeredintratumorally at a dose that is up to 100-fold lower than a dose givenby systemic administration. In certain embodiments, the anti-CTLA-4antibody or pharmaceutical composition described herein is administered(e.g., intratumorally or systemically) as a monotherapy. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered intratumorally and the method furthercomprises administering an additional therapeutic agent to the subject.In certain embodiments, the additional therapeutic agent is administeredsystemically. In certain embodiments, the subject has a solid tumor andthe additional therapeutic agent is an anti-PD-1 antibody. In certainembodiments, the anti-PD-1 antibody is pembrolizumab or nivolumab. Incertain embodiments, the pembrolizumab is administered at a dose of 200mg every three weeks. In certain embodiments, the subject has head andneck squamous cell carcinoma and the additional therapeutic agent is ananti-EGFR antibody. In certain embodiments, the anti-EGFR antibody iscetuximab. In certain embodiments, the subject has HER2+ breast cancerand the additional therapeutic agent is an anti-HER2 antibody. Incertain embodiments, the anti-HER2 antibody is trastuzumab. In certainembodiments, these methods further comprise administering achemotherapeutic agent to the subject. In certain embodiments, thechemotherapeutic agent is administered systemically. In certainembodiments, the chemotherapeutic agent is gemcitabine. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered intratumorally and the subject has anadvanced or metastatic solid tumor. In certain embodiments, theanti-CTLA-4 antibody or pharmaceutical composition described herein isadministered intratumorally and the subject has head and neck cancer(e.g., relapsed/refractory head and neck squamous cell carcinoma). Incertain embodiments, the anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered intratumorally and thesubject has breast cancer (e.g., relapsed/refractory HER2+ breastcancer). In certain embodiments, the anti-CTLA-4 antibody orpharmaceutical composition described herein is delivered to a tumordraining lymph node. In certain embodiments, the anti-CTLA-4 antibody orpharmaceutical composition described herein is delivered via a localizedadministration (e.g., subcutaneous administration). In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is delivered via a localized administration (e.g.,subcutaneous administration) at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3mg/kg, 1 mg/kg, or 3 mg/kg. In certain embodiments, the anti-CTLA-4antibody or pharmaceutical composition described herein is delivered viaa localized administration (e.g., subcutaneous administration) at a dosethat is up to 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or 200-fold lower than adose given by systemic administration. In certain embodiments, theanti-CTLA-4 antibody or pharmaceutical composition described herein isdelivered via a localized administration (e.g., subcutaneousadministration) at a dose that is up to 10-fold lower than a dose givenby systemic administration. In certain embodiments, the anti-CTLA-4antibody or pharmaceutical composition described herein is delivered viaa localized administration (e.g., subcutaneous administration) at a dosethat is up to 100-fold lower than a dose given by systemicadministration. In certain embodiments, the anti-CTLA-4 antibody orpharmaceutical composition described herein is delivered via a localizedadministration (e.g., subcutaneous administration) and the methodfurther comprises administering an additional therapeutic agent to thesubject. In certain embodiments, the additional therapeutic agent is avaccine. In certain embodiments, the vaccine comprises a heat shockprotein peptide complex (HSPPC) comprising a heat shock proteincomplexed with an antigenic peptide. In one embodiment, the heat shockprotein is gp96 protein and is complexed with a tumor-associatedantigenic peptide, wherein the HSPPC is derived from a tumor obtainedfrom a subject. In certain embodiments, the heat shock protein isselected from the group consisting of hsc70, hsp70, hsp90, hsp110,grp170, gp96, calreticulin, a mutant thereof, and combinations of two ormore thereof. In certain embodiments, the heat shock protein is hsc70.In certain embodiments, the heat shock protein is hsp70. In certainembodiments, the antigenic peptide is synthetic. In certain embodiments,the subject has cancer. In certain embodiments, the subject has aninfectious disease. In certain embodiments, these methods furthercomprise administering an additional therapeutic agent to the subject.In certain embodiments, the additional therapeutic agent is achemotherapeutic or a checkpoint targeting agent. In certainembodiments, the checkpoint targeting agent is selected from the groupconsisting of an antagonist anti-PD-1 antibody, an antagonist anti-PD-L1antibody, an antagonist anti-PD-L2 antibody, an antagonist anti-CTLA-4antibody, an antagonist anti-TIM-3 antibody, an antagonist anti-LAG-3antibody, an antagonist anti-CEACAM1 antibody, an agonist anti-GITRantibody, an agonist anti-OX40 antibody, and an agonist anti-CD137antibody, an agonist anti-DR3 antibody, an agonist anti-TNFSF14antibody, and an agonist anti-CD27 antibody. In certain embodiments, theadditional therapeutic agent is radiotherapy. In certain embodiments,the additional therapeutic agent is an inhibitor ofindoleamine-2,3-dioxygenase (IDO). Suitable IDO inhibitors include,without limitation, epacadostat, F001287, indoximod, and NLG919. Incertain embodiments, the additional therapeutic agent is a vaccine. Incertain embodiments, the vaccine comprises a heat shock protein peptidecomplex (HSPPC) comprising a heat shock protein complexed with anantigenic peptide. In one embodiment, the heat shock protein is gp96protein and is complexed with a tumor-associated antigenic peptide,wherein the HSPPC is derived from a tumor obtained from a subject.

In another aspect, the instant disclosure provides a method of treatingan infectious disease in a subject, the method comprising administeringto the subject an effective amount of an anti-CTLA-4 antibody orpharmaceutical composition described herein. In another aspect, theinstant disclosure provides a method of preventing an infectious diseasein a subject, the method comprising administering to the subject aneffective amount of an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein.

In one embodiment, the present invention relates to an antibody of thepresent invention, a polynucleotide of the invention, a vector of theinvention, and/or a recombinant host cell of the invention, for use as amedicament.

In one embodiment, the present invention relates to an antibody of thepresent invention, a polynucleotide of the invention, a vector of theinvention, and/or a recombinant host cell of the invention, for use as adiagnostic.

In one embodiment, the present invention relates to the use of anantibody of the present invention, a polynucleotide of the invention, avector of the invention, and/or a recombinant host cell of theinvention, for the in vitro detection of human CTLA-4 in a biologicalsample.

In one aspect, provided herein is a pharmaceutical compositioncomprising an anti-CTLA-4 antibody described herein and apharmaceutically acceptable carrier or excipient, for use as amedicament.

In one aspect, provided herein is a pharmaceutical compositioncomprising an anti-CTLA-4 antibody described herein and apharmaceutically acceptable carrier or excipient, for use as adiagnostic.

In one aspect, provided herein is a pharmaceutical compositioncomprising an anti-CTLA-4 antibody described herein, a polynucleotide ofthe invention, a vector of the invention, and/or a recombinant host cellof the invention, and a pharmaceutically acceptable carrier orexcipient. In one aspect, the pharmaceutical composition is for use as amedicament and/or diagnostic.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for use in a method for increasingT-cell activation in response to an antigen.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for use in a method for increasingT-cell activation in response to an antigen in a subject.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for use in a method for increasingT-cell activation in response to an antigen in a subject comprisingadministering to the subject an effective amount of an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the invention.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for use in a method for thetreatment of cancer.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for use in a method for thetreatment of cancer in a subject.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for use in a method for thetreatment of cancer in a subject comprising administering to the subjectan effective amount of an antibody, polynucleotide, vector, recombinanthost cell, and/or pharmaceutical composition of the invention.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent, preferably an anti-PD-1 antibody, for use as a medicament.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent, preferably an anti-PD-1 antibody, for use in a method for thetreatment of cancer. In a preferred embodiment, the cancer is a solidtumor. In another preferred embodiment, the antibody, polynucleotide,vector, recombinant host cell, and/or pharmaceutical composition of thepresent invention is administered intratumorally to the subject,preferably administered intratumorally to the subject at 0.01 mg/kg,0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg, optionally at aninterval of once every three weeks.

In one aspect, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent, preferably an anti-PD-1 antibody.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an anti-EGFR antibody, andoptionally (c) a chemotherapeutic agent, for use as a medicament.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an anti-EGFR antibody, andoptionally (c) a chemotherapeutic agent, for use in a method for thetreatment of cancer. In a preferred embodiment, the cancer is head andneck squamous cell carcinoma. In another preferred embodiment, theantibody, polynucleotide, vector, recombinant host cell, and/orpharmaceutical composition of the present invention is administeredintratumorally to the subject, preferably administered intratumorally tothe subject at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or3 mg/kg, optionally at an interval of once every three weeks.

In one aspect, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an anti-EGFR antibody, andoptionally (c) a chemotherapeutic agent.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an anti-HER2 antibody, andoptionally (c) a chemotherapeutic agent, for use as a medicament.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an anti-HER2 antibody, andoptionally (c) a chemotherapeutic agent, for use in a method for thetreatment of HER2+ breast cancer. In another preferred embodiment, theantibody, polynucleotide, vector, recombinant host cell, and/orpharmaceutical composition of the present invention is administeredintratumorally to the subject, preferably administered intratumorally tothe subject at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or3 mg/kg, optionally at an interval of once every three weeks.

In one aspect, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an anti-HER2 antibody, andoptionally (c) a chemotherapeutic agent.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for use in a method for thetreatment of cancer, wherein the antibody, polynucleotide, vector,recombinant host cell, and/or pharmaceutical composition of the presentinvention is administered intratumorally to the subject, preferablyadministered intratumorally to the subject at 0.01 mg/kg, 0.03 mg/kg,0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, or 3 mg/kg, optionally at an interval ofonce every three weeks.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for use in a method for thetreatment of cancer, wherein the antibody, polynucleotide, vector,recombinant host cell, and/or pharmaceutical composition of the presentinvention is administered subcutaneously or intravenously to thesubject, preferably administered intravenously to the subject at 0.01mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, 6 mg/kg, or10 mg/kg, optionally at an interval of once every three weeks.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent, for use as a medicament. In a preferred embodiment, theadditional therapeutic agent is a chemotherapeutic agent or a checkpointtargeting agent or an inhibitor of indoleamine-2,3-dioxygenase (IDO) ora vaccine.

In one aspect, the present invention relates to (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent, for use in a method for the treatment of cancer. In a preferredembodiment, the additional therapeutic agent is a chemotherapeutic agentor a checkpoint targeting agent or an inhibitor ofindoleamine-2,3-dioxygenase (IDO) or a vaccine.

In one aspect, the present invention relates to a pharmaceuticalcomposition, kit or kit-of-parts comprising (a) an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention and (b) an additional therapeuticagent. In a preferred embodiment, the additional therapeutic agent is achemotherapeutic agent or a checkpoint targeting agent or an inhibitorof indoleamine-2,3-dioxygenase (IDO) or a vaccine.

In one aspect, the present invention relates to an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for use in a method for thetreatment of cancer, and/or for use in a method for increasing T-cellactivation in response to an antigen, wherein the antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention is delivered to a tumor draininglymph node.

In one aspect, the present invention relates to the use of an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention in a method for the treatment ofcancer, and/or in a method for increasing T-cell activation in responseto an antigen in a subject, wherein the antibody, polynucleotide,vector, recombinant host cell, and/or pharmaceutical composition of thepresent invention is delivered to a tumor draining lymph node.

In one aspect, the present invention relates to the use of an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for preparing medicaments forimmunotherapy, for example, for increasing T-cell activation in responseto an antigen in a subject, treating cancer, or treating or preventinginfectious diseases.

In one aspect, the present invention relates to the use of an antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention, for preparing medicaments forimmunotherapy, for example, for increasing T-cell activation in responseto an antigen in a subject, treating cancer, or treating or preventinginfectious diseases, wherein the antibody, polynucleotide, vector,recombinant host cell, and/or pharmaceutical composition of the presentinvention is delivered to a tumor draining lymph node.

In one aspect, the present invention relates to the use of (a) anantibody, polynucleotide, vector, recombinant host cell, and/orpharmaceutical composition of the present invention and (b) an anti-HER2antibody, and optionally (c) a chemotherapeutic agent, to prepare amedicament for immunotherapy, for example, for increasing T-cellactivation in response to an antigen in a subject, treating cancer, ortreating or preventing infectious diseases.

In one aspect, the present invention relates to the use of (a) anantibody, polynucleotide, vector, recombinant host cell, and/orpharmaceutical composition of the present invention and (b) an anti-HER2antibody, and optionally (c) a chemotherapeutic agent, to prepare amedicament for immunotherapy, for example, for increasing T-cellactivation in response to an antigen in a subject, treating cancer, ortreating or preventing infectious diseases, wherein the antibody,polynucleotide, vector, recombinant host cell, and/or pharmaceuticalcomposition of the present invention is delivered to a tumor draininglymph node.

5. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D, 1E, 1F, and 1G are flow cytometry histogramsshowing the binding of anti-CTLA-4 antibodies or an IgG₁ isotype controlantibody to Jurkat cells engineered to express human CTLA-4 on the cellsurface. The anti-CTLA-4 antibodies tested are: AGEN1884.H1.1 (IgG₁),AGEN1884.H1.2 (IgG₁), AGEN1884.H1.3 (IgG₁), AGEN1884.H2.1 (IgG₁),AGEN1884.H2.2 (IgG₁), AGEN1884.H2.3 (IgG₁), and AGEN1884.H3 (IgG₁).

FIG. 2 is a graph showing IL-2 production of primary human PBMCsfollowing incubation under sub-optimal stimulation with theStaphylococcal Enterotoxin A (SEA) superantigen in the absence orpresence of the anti-CTLA-4 antibody AGEN1884.H3 (IgG₁) or an isotypecontrol antibody (IgG₁). Replicates of eight were performed for eachgroup and the mean values of the eight replicates are indicated with ablack bar.

FIG. 3 is a graph showing results from an IL-2-luciferase reporter assaydemonstrating that blockade of CTLA-4 leads to T cell activation. Foldresponse of luciferase expression, a surrogate marker for IL-2 geneactivation, is plotted over a range of antibody concentrations forAGEN1884.H3 (IgG₁) or an isotype control antibody (IgG₁).

FIG. 4 is a graph showing results from a reporter assay wheresimultaneous engagement of AGEN1884.H3 (IgG₁) to target cells (viaCTLA-4 binding) and effector cells (via FcγRIIIA binding) triggersexpression of luciferase by the effector cell line. Luciferase activityis a surrogate marker for FcγRIIIA signaling. Fold response of RLUvalues is plotted against a range of antibody concentrations forAGEN1884.H3 (IgG₁) and an isotype control antibody (IgG₁).

FIGS. 5A, 5B, 5C, and 5D are flow cytometry histograms showingCTLA-4-expressing Jurkat cells incubated with the anti-CTLA-4 antibodyAGEN1884.H3 (IgG₁), AGEN1884.H3 (IgG₁ S239D/I332E), AGEN1884.H3 (IgG₁S239D/A330L/I332E), or AGEN1884.H3 (IgG₁ L235V/F243L/R292P/Y300L/P396L),or an isotype control antibody. Antibody binding was detected using afluorochrome-conjugated secondary antibody.

FIGS. 6A and 6B are graphs showing blocking of binding between humanCTLA-4 and its ligands, CD80 and CD86, respectively, by AGEN1884.H3(IgG₁ S239D/A330L/I332E). Jurkat cells engineered to constitutivelyexpress human CTLA-4 were incubated with anti-CTLA-4 antibodyAGEN1884.H3 (IgG₁-S239D/A330E/I332E), a reference anti-CTLA-4 antibody,or an isotype control antibody (IgG₁), and then stained with theindicated fluorescently labeled ligand. Ligand binding was then assessedby flow cytometry.

FIGS. 7A, 7B, and 7C are graphs showing IL-2 production of primary humanPBMCs cultured under sub-optimal stimulation with the SEA superantigenin the absence or presence of an isotype control antibody (IgG₁) or ananti-CTLA-4 antibody. FIGS. 7A and 7B are graphs showing IL-2 productionstimulated by either a single dose or a dose titration of the isotypecontrol antibody (IgG₁) or the anti-CTLA-4 antibodies AGEN1884.H3(IgG₁), AGEN1884.H3 (IgG₁ S239D/I332E), AGEN1884.H3 (IgG₁S239D/A330L/I332E), and AGEN1884.H3 (IgG₁L235V/F243L/R292P/Y300L/P396L). In the study shown in FIG. 7B, inaddition to the isotype control antibody (IgG₁) or the anti-CTLA-4antibody, the cells in each sample were also incubated with an IgG₄S228P isotype control antibody. FIG. 7C is a graph showing IL-2production stimulated by a dose titration of the isotype controlantibody (IgG₁) or the anti-CTLA-4 antibodies AGEN1884 (IgG₁), AGEN1884(IgG₁ S239D/I332E), AGEN1884 (IgG₁ S239D/A330L/I332E), and afucosylatedAGEN1884 (IgG₁).

FIG. 8A is an immunoblot analysis for phosphorylated ZAP70 (Y493) inhuman PBMCs following stimulation with 50 ng/ml of SEA superantigen and10 μg/ml of isotype control antibody (IgG₁) or the anti-CTLA-4antibodies AGEN1884.H3 (IgG₁), AGEN1884.H3 (IgG₁ S239D/A330L/I332E), orAGEN1884.H3 (IgG₁ N297A). FIG. 8B is a chart showing normalizeddensitometric analysis of the data shown in FIG. 8A.

FIGS. 9A, 9B, 9C, and 9D are graphs showing antitumor efficacy andintratumoral regulatory T cell (Treg) depletion induced by Fc variantsof murine anti-CTLA-4 antibody 9D9. FIG. 9A shows tumor growth in CT26mice following single-dose treatment with murine anti-CTLA-4 antibody9D9 (mIgG2a), an Fc-silent variant of anti-CTLA-4 antibody 9D9(mIgG2a-N297A), an Fc variant of anti-CTLA-4 antibody 9D9(mIgG2a-S239D/A330L/I332E), or an isotype control antibody (mIgG2a). Theupper panel shows median tumor volume over time for each treatmentgroup. The remaining panels show tumor volume over time for individualanimals in each treatment group. FIG. 9B shows the effect of anti-CTLA-4antibody treatment on T cell populations from tumor infiltratescollected from mice treated with single doses of anti-CTLA-4 antibody9D9 (mIgG2a), anti-CTLA-4 antibody 9D9 (mIgG2a-N297A), anti-CTLA-4antibody 9D9 (mIgG2a-S239D/A330L/I332E), or isotype control antibody(mIgG2a). Tumor infiltrates were harvested and analyzed by flowcytometry at indicated time points after injection with antibody. Cellpopulations analyzed include: FoxP3+ Tregs (upper left panel), CD45+leukocytes (upper right panel), and CD4+ non-Tregs (lower left panel).The lower right panel shows the ratio of CD8+ T cells to Tregs observedin tumor infiltrates. FIG. 9C shows FoxP3+ Treg populations over time intumor-draining lymph nodes harvested from mice treated as described forFIG. 9B. FIG. 9D shows fold-change in splenic FoxP3+ Tregs at 72 hoursafter treatment as described for FIG. 9B.

FIG. 10 is a series of graphs showing antitumor efficacy of murineanti-CTLA-4 antibodies when combined with tumor vaccine derived from aHPV+ tumor (viral antigens E6/E7). Shown are tumor volume over time forindividual mice receiving no treatment, isotype control antibody(mIgG2a), anti-CTLA-4 antibody 9D9 (mIgG2a), or an Fc variant ofanti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E). Graphs in the toprow show results for animals that were administered the indicatedantibody treatment only. Graphs in the bottom row show results foranimals that were administered the indicated antibody treatment incombination with tumor vaccine.

FIGS. 11A and 11B are graphs showing gene expression and CpG methylationof human T cell populations. CD4⁺ CD25^(+/−) FOXP3⁻ non-regulatory Tcells (Teff) and CD4⁺ CD25⁺ FOXP3⁺ regulatory T cells (Treg) wereisolated from peripheral blood of healthy donors, expanded, andactivated. FIG. 11A shows FOXP3, intracellular CTLA-4, and membraneCTLA-4 levels in each activated T cell population, as determined by flowcytometry. FIG. 11B shows the level of CpG methylation in CpG regionswithin the FOXP3 (top panel) and CTLA4 (bottom panel) loci in naïve Tcells, activated effector T cells, and activated regulatory T cells,each from the same donor.

FIGS. 12A and 12B are graphs showing time courses of antibody dependentcellular cytotoxicity (ADCC) of human CTLA-4+ target cells afterincubation with anti-CTLA-4 antibody AGEN1884.H3 (IgG₁) or Fc variantsthereof. NK-92 cells (FcγRIIIA V158-expressing) were co-cultured withCTLA-4+ target cells that were incubated with different Fc variants ofanti-CTLA-4 antibodies or an IgG₁ isotype control (10 μg/ml). Highcontent microscopy of caspase 3/7 activation was then used to quantifyADCC activity. FIG. 12A shows ADCC activity in Jurkat cells engineeredto express CTLA-4 on the cell surface, when incubated with AGEN1884.H3(IgG₁), AGEN1884.H3 (IgG₁ N297A), AGEN1884.H3 (IgG₁ S239D/A330L/I332E),AGEN1884.H3 (IgG₁ S267E/L328F), afucosylated AGEN1884.H3 (IgG₁), or anisotype control antibody (IgG₁). FIG. 12B shows ADCC activity in primaryhuman activated effector T cells (left panel) or regulatory T cells(right panel) when incubated with these antibodies.

FIGS. 13A, 13B, 13C, and 13D are graphs showing the effects ofanti-CTLA-4 antibody variants on T cell function when administered aloneor in combination with an anti-PD-1 antibody. Human PBMCs were isolatedfrom two donors and incubated under stimulatory culture conditions withanti-CTLA-4 antibody AGEN1884.H3 (IgG₁), an Fc variant anti-CTLA-4antibody AGEN1884.H3 (IgG₁ S239D/A330L/I332E), or an isotype controlantibody (IgG₁), in combination with a reference anti-PD-1 antibody oran isotype control antibody (IgG₄), as indicated. For each treatmentcondition listed, a dosage titration was used for the first-listedantibody, and a fixed concentration (5 μg/ml) was used for thesecond-listed antibody. This experiment was performed twice, for a totalof two replicates per donor. The levels of IL-2 production induced byeach antibody combination on PBMCs collected from the first donor areshown in FIG. 13A (replicate 1) and FIG. 13B (replicate 2). The levelsof IL-2 production induced by each antibody combination on PBMCscollected from the second donor are shown in FIG. 13C (replicate 1) andFIG. 13D (replicate 2).

FIGS. 14A, 14B, and 14C are a series of sequence alignments. FIG. 14A isa sequence alignment for human CTLA-4 (SEQ ID NO: 33), cynomolgus monkeyCTLA-4 (SEQ ID NO: 40), mouse CTLA-4 (SEQ ID NO: 41), and rat CTLA-4(SEQ ID NO: 42). Dots represent residues identical to correspondinghuman residues. An “*” (asterisk) indicates positions which have asingle, fully conserved residue. A “:” (colon) indicates conservationbetween groups of strongly similar properties. A “.” (period) indicatesconservation between groups of weakly similar properties. FIGS. 14B and14C are sequence alignments for human CTLA-4 (residues 1-144 andresidues 145-223 of SEQ ID NO: 33, respectively), cynomolgus monkeyCTLA-4 (residues 1-144 and residues 145-223 of SEQ ID NO: 40,respectively), human CD28 (residues 1-127 and residues 128-220 of SEQ IDNO: 43, respectively), human ICOS (residues 1-124 and residues 125-199of SEQ ID NO: 44, respectively), human BTLA (residues 1-125 and residues126-289 of SEQ ID NO: 45, respectively), and human PD-1 (residues 1-143and residues 144-288 of SEQ ID NO: 46, respectively). The two regionsshowing strong decrease in deuterium uptake when human CTLA-4 was boundto AGEN1884-Fab are underlined in FIGS. 14A-14C: residues 80-82 (QVT,SEQ ID NO: 39) and residues 135-149 (YPPPYYLGIGNGTQI, SEQ ID NO: 37),numbered according to SEQ ID NO: 33.

6. DETAILED DESCRIPTION

The instant disclosure provides antibodies that specifically bind toCTLA-4 (e.g., human CTLA-4) and antagonize CTLA-4 function, e.g.,CTLA-4-mediated immune suppression. Also provided are pharmaceuticalcompositions comprising these antibodies, nucleic acids encoding theseantibodies, expression vectors and host cells for making theseantibodies, and methods of treating a subject using these antibodies.The antibodies described herein are particularly useful for increasing Tcell activation in response to an antigen (e.g., a tumor antigen or aninfectious disease antigen), and hence for treating cancer in a subjector treating or preventing an infectious disease in a subject. Allinstances of “isolated antibodies” described herein are additionallycontemplated as antibodies that may be, but need not be, isolated. Allinstances of “isolated polynucleotides” described herein areadditionally contemplated as polynucleotides that may be, but need notbe, isolated. All instances of “antibodies” described herein areadditionally contemplated as antibodies that may be, but need not be,isolated. All instances of “polynucleotides” described herein areadditionally contemplated as polynucleotides that may be, but need notbe, isolated.

The skilled worker will appreciate that a glutamate (E) or glutamine (Q)amino acid residue at the N-terminus of a heavy chain variable regionand/or a light chain variable region of any one of the antibodiesdescribed herein (e.g., an anti-CTLA4 antibody) can, under certainconditions, spontaneously convert to pyroglutamate by post-translationalcyclization of the free amino group to form a lactam. Accordingly, incertain embodiments of each and every one of the methods, uses,pharmaceutical compositions, or kits described herein, the N-terminalamino acid residue of one or more heavy chain variable regions and/orlight chain variable regions of the antibody has been converted topyroglutamate (e.g., as a result of post-translational cyclization ofthe free amino group of the N-terminal E or Q residue).

6.1 Definitions

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above (e.g., up to 5% to 10% above) and 5% to 10% below (e.g., upto 5% to 10% below) the value or range remain within the intendedmeaning of the recited value or range.

As used herein, the term “CTLA-4” refers to cytotoxicT-lymphocyte-associated protein 4. As used herein, the term “humanCTLA-4” refers to a human CTLA-4 protein encoded by a wild type humanCTLA-4 gene, e.g., GenBank™ accession number NM_005214.4 orNM_001037631.2. An exemplary immature amino acid sequence of humanCTLA-4 is provided as SEQ ID NO: 33.

As used herein, the terms “antibody” and “antibodies” include fulllength antibodies, antigen-binding fragments of full length antibodies,and molecules comprising antibody CDRs, VH regions or VL regions.Examples of antibodies include monoclonal antibodies, recombinantlyproduced antibodies, monospecific antibodies, multispecific antibodies(including bispecific antibodies), human antibodies, humanizedantibodies, chimeric antibodies, immunoglobulins, synthetic antibodies,tetrameric antibodies comprising two heavy chain and two light chainmolecules, an antibody light chain monomer, an antibody heavy chainmonomer, an antibody light chain dimer, an antibody heavy chain dimer,an antibody light chain-antibody heavy chain pair, intrabodies,heteroconjugate antibodies, antibody-drug conjugates, single domainantibodies, monovalent antibodies, single chain antibodies orsingle-chain Fvs (scFv), camelized antibodies, affybodies, Fabfragments, F(ab′)₂ fragments, disulfide-linked Fvs (sdFv),anti-idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Idantibodies), and antigen-binding fragments of any of the above. Incertain embodiments, antibodies described herein refer to polyclonalantibody populations. Antibodies can be of any type (e.g., IgG, IgE,IgM, IgD, IgA or IgY), any class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁ orIgA₂), or any subclass (e.g., IgG_(2a) or IgG_(2b)) of immunoglobulinmolecule. In certain embodiments, antibodies described herein are IgGantibodies, or a class (e.g., human IgG₁ or IgG₄) or subclass thereof.In a specific embodiment, the antibody is a humanized monoclonalantibody. In another specific embodiment, the antibody is a humanmonoclonal antibody.

As used herein, the terms “VH region” and “VL region” refer to singleantibody heavy and light chain variable regions, respectively,comprising FR (Framework Regions) 1, 2, 3 and 4 and CDR (ComplementarityDetermining Regions) 1, 2 and 3 (see Kabat et al., (1991) Sequences ofProteins of Immunological Interest (NIH Publication No. 91-3242,Bethesda), which is herein incorporated by reference in its entirety).

As used herein, the term “CDR” or “complementarity determining region”means the noncontiguous antigen combining sites found within thevariable region of both heavy and light chain polypeptides. Theseparticular regions have been described by Kabat et al., J. Biol. Chem.252, 6609-6616 (1977) and Kabat et al., Sequences of protein ofimmunological interest. (1991), by Chothia et al., J. Mol. Biol.196:901-917 (1987), and by MacCallum et al., J. Mol. Biol. 262:732-745(1996), all of which are herein incorporated by reference in theirentireties, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. In certainembodiments, the term “CDR” is a CDR as defined by Kabat et al., J.Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of proteinof immunological interest. (1991). In certain embodiments, the term“CDR” is a CDR as defined by Chothia et al., J. Mol. Biol. 196:901-917(1987). In certain embodiments, the term “CDR” is a CDR as defined byMacCallum et al., J. Mol. Biol. 262:732-745 (1996) and Martin A.“Protein Sequence and Structure Analysis of Antibody Variable Domains,”in Antibody Engineering, Kontermann and Dübel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001).

As used herein, the term “framework (FR) amino acid residues” refers tothose amino acids in the framework region of an immunoglobulin chain.The term “framework region” or “FR region” as used herein, includes theamino acid residues that are part of the variable region, but are notpart of the CDRs (e.g., using the Kabat or Chothia definition of CDRs).

As used herein, the terms “variable region” and “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 125amino acids in the mature heavy chain and about 90 to 115 amino acids inthe mature light chain, which differ extensively in sequence amongantibodies and are used in the binding and specificity of a particularantibody for its particular antigen. The variability in sequence isconcentrated in those regions called complementarity determining regions(CDRs) while the more highly conserved regions in the variable domainare called framework regions (FR). Without wishing to be bound by anyparticular mechanism or theory, it is believed that the CDRs of thelight and heavy chains are primarily responsible for the interaction andspecificity of the antibody with antigen. In certain embodiments, thevariable region is a human variable region. In certain embodiments, thevariable region comprises rodent or murine CDRs and human frameworkregions (FRs). In particular embodiments, the variable region is aprimate (e.g., non-human primate) variable region. In certainembodiments, the variable region comprises rodent or murine CDRs andprimate (e.g., non-human primate) framework regions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

As used herein, the terms “constant region” and “constant domain” areinterchangeable and are common in the art. The constant region is anantibody portion, e.g., a carboxyl terminal portion of a light and/orheavy chain which is not directly involved in binding of an antibody toantigen but which can exhibit various effector functions, such asinteraction with an Fc receptor (e.g., Fc gamma receptor). The constantregion of an immunoglobulin molecule generally has a more conservedamino acid sequence relative to an immunoglobulin variable domain.

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG, and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃, and IgG₄.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art.

As used herein, the term “EU numbering system” refers to the EUnumbering convention for the constant regions of an antibody, asdescribed in Edelman, G. M. et al., Proc. Natl. Acad. USA, 63, 78-85(1969) and Kabat et al., Sequences of Proteins of ImmunologicalInterest, U.S. Dept. Health and Human Services, 5th edition, 1991, eachof which is herein incorporated by reference in its entirety.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured and/or expressedin a number of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D)), and equilibrium associationconstant (K_(A)). The K_(D) is calculated from the quotient ofk_(off)/k_(on), whereas K_(A) is calculated from the quotient ofk_(on)/k_(off). k_(on) refers to the association rate constant of, e.g.,an antibody to an antigen, and k_(off) refers to the dissociation rateconstant of, e.g., an antibody to an antigen. The k_(on) and k_(off) canbe determined by techniques known to one of ordinary skill in the art,such as BIAcore® or KinExA. As used herein, a “lower affinity” refers toa larger K_(D).

As used herein, the terms “specifically binds,” “specificallyrecognizes,” “immunospecifically binds,” and “immunospecificallyrecognizes” are analogous terms in the context of antibodies and referto molecules that bind to an antigen (e.g., epitope or immune complex)as such binding is understood by one skilled in the art. For example, amolecule that specifically binds to an antigen can bind to otherpeptides or polypeptides, generally with lower affinity as determinedby, e.g., immunoassays, BIAcore®, KinExA 3000 instrument (SapidyneInstruments, Boise, Id.), or other assays known in the art. In aspecific embodiment, molecules that specifically bind to an antigen bindto the antigen with a K_(A) that is at least 2 logs (i.e., factors of10), 2.5 logs, 3 logs, 4 logs or greater than the K_(A) when themolecules bind non-specifically to another antigen.

In another specific embodiment, molecules that specifically bind to anantigen do not cross react with other proteins under similar bindingconditions. In another specific embodiment, molecules that specificallybind to CTLA-4 do not cross react with other non-CTLA-4 proteins. In aspecific embodiment, provided herein is an antibody that binds to CTLA-4(e.g., human CTLA-4) with higher affinity than to another unrelatedantigen. In certain embodiments, provided herein is an antibody thatbinds to CTLA-4 (e.g., human CTLA-4) with a 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or higher affinitythan to another, unrelated antigen as measured by, e.g., aradioimmunoassay, surface plasmon resonance, or kinetic exclusion assay.In a specific embodiment, the extent of binding of an anti-CTLA-4antibody described herein to an unrelated, non-CTLA-4 protein is lessthan 10%, 15%, or 20% of the binding of the antibody to CTLA-4 proteinas measured by, e.g., a radioimmunoassay.

As used herein, the term “afucosylation” or “afucosylated” in thecontext of an Fc refers to a substantial lack of a fucose covalentlyattached, directly or indirectly, to residue 297 of the human IgG₁ Fcregion, numbered according to the EU numbering system, or thecorresponding residue in non-IgG₁ or non-human IgG₁ immunoglobulins.Thus, in a composition comprising a plurality of afucosylatedantibodies, at least 70% of the antibodies will not be fucosylated,directly or indirectly (e.g., via intervening sugars) at residue 297 ofthe Fc region of the antibodies, and in some embodiments at least 80%,85%, 90%, 95%, or 99% will not be fucosylated, directly or indirectly,at residue 297 of the Fc region.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody can specificallybind. An epitope can be, for example, contiguous amino acids of apolypeptide (linear or contiguous epitope) or an epitope can, forexample, come together from two or more non-contiguous regions of apolypeptide or polypeptides (conformational, non-linear, discontinuous,or non-contiguous epitope). In certain embodiments, the epitope to whichan antibody binds can be determined by, e.g., NMR spectroscopy, X-raydiffraction crystallography studies, ELISA assays, hydrogen/deuteriumexchange coupled with mass spectrometry (e.g., liquid chromatographyelectrospray mass spectrometry), array-based oligo-peptide scanningassays (e.g., constraining peptides using CLIPS (Chemical Linkage ofPeptides onto Scaffolds) to map discontinuous or conformationalepitopes), and/or mutagenesis mapping (e.g., site-directed mutagenesismapping). For X-ray crystallography, crystallization may be accomplishedusing any of the known methods in the art (e.g., Giegé R et al., (1994)Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A(1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of whichare herein incorporated by reference in their entireties).Antibody:antigen crystals may be studied using well known X-raydiffraction techniques and may be refined using computer software suchas X-PLOR (Yale University, 1992, distributed by Molecular Simulations,Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff H Wet al.; U.S. 2004/0014194), and BUSTER (Bricogne G (1993) ActaCrystallogr D Biol Crystallogr 49(Pt 1): 37-60; Bricogne G (1997) MethEnzymol 276A: 361-423, ed Carter C W; Roversi P et al., (2000) ActaCrystallogr D Biol Crystallogr 56(Pt 10): 1316-1323), all of which areherein incorporated by reference in their entireties. Mutagenesismapping studies may be accomplished using any method known to one ofskill in the art. See, e.g., Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham B C & Wells J A (1989) Science 244: 1081-1085,each of which is herein incorporated by reference in its entirety, for adescription of mutagenesis techniques, including alanine scanningmutagenesis techniques. CLIPS (Chemical Linkage of Peptides ontoScaffolds) is a technology to present one or more peptides in astructurally constrained configuration to behave as functional mimics ofcomplex protein domains. See, e.g., U.S. Publication Nos. US2008/0139407 A1 and US 2007/099240 A1, and U.S. Pat. No. 7,972,993, eachof which is herein incorporated by reference in its entirety. In aspecific embodiment, the epitope of an antibody is determined usingalanine scanning mutagenesis studies. In a specific embodiment, theepitope of an antibody is determined using hydrogen/deuterium exchangecoupled with mass spectrometry. In a specific embodiment, the epitope ofan antibody is determined using CLIPS Epitope Mapping Technology fromPepscan Therapeutics.

As used herein, the term “an epitope located within a region of humanCTLA-4” consisting of a particular amino acid sequence or a set of aminoacid residues refers to an epitope comprising one or more of the aminoacid residues of the specified region, wherein the specified regionincludes the first specified amino acid residue and the last specifiedamino acid residue of the region of human CTLA-4. In certainembodiments, the epitope comprises each one of the amino acid residueslocated within the specified region. In certain embodiments, one or moreadditional amino acid residues of human CTLA-4 outside the specifiedregion bind to an antibody together with an epitope located within thespecified region.

As used herein, the terms “T cell receptor” and “TCR” are usedinterchangeably and refer to full length heterodimeric αβ or γδ TCRs,antigen-binding fragments of full length TCRs, and molecules comprisingTCR CDRs or variable regions. Examples of TCRs include, but are notlimited to, full length TCRs, antigen-binding fragments of full lengthTCRs, soluble TCRs lacking transmembrane and cytoplasmic regions,single-chain TCRs containing variable regions of TCRs attached by aflexible linker, TCR chains linked by an engineered disulfide bond,monospecific TCRs, multi-specific TCRs (including bispecific TCRs), TCRfusions, human TCRs, humanized TCRs, chimeric TCRs, recombinantlyproduced TCRs, and synthetic TCRs. The term encompasses wild-type TCRsand genetically engineered TCRs (e.g., a chimeric TCR comprising achimeric TCR chain which includes a first portion from a TCR of a firstspecies and a second portion from a TCR of a second species).

As used herein, the terms “major histocompatibility complex” and “MHC”are used interchangeably and refer to an MHC class I molecule and/or anMHC class II molecule.

As used herein, the term “peptide-MHC complex” refers to an MHC molecule(MHC class I or MHC class II) with a peptide bound in the art-recognizedpeptide binding pocket of the MHC.

As used herein, the term “treat,” “treating,” and “treatment” refer totherapeutic or preventative measures described herein. The methods of“treatment” employ administration of an antibody to a subject having adisease or disorder, or predisposed to having such a disease ordisorder, in order to prevent, cure, delay, reduce the severity of, orameliorate one or more symptoms of the disease or disorder or recurringdisease or disorder, or in order to prolong the survival of a subjectbeyond that expected in the absence of such treatment.

As used herein, the term “effective amount” in the context of theadministration of a therapy to a subject refers to the amount of atherapy that achieves a desired prophylactic or therapeutic effect.

As used herein with respect to the response of a cancer to a therapy,the terms “refractory” and “resistant” have their art-recognized meaningand are used interchangeably.

As used herein, the term “subject” includes any human or non-humananimal. In one embodiment, the subject is a human or non-human mammal.In one embodiment, the subject is a human.

The determination of “percent identity” between two sequences (e.g.,amino acid sequences or nucleic acid sequences) can be accomplishedusing a mathematical algorithm. A specific, non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin S & Altschul S F (1990) PNAS 87: 2264-2268,modified as in Karlin S & Altschul S F (1993) PNAS 90: 5873-5877, eachof which is herein incorporated by reference in its entirety. Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul S F et al., (1990) J Mol Biol 215: 403, which is hereinincorporated by reference in its entirety. BLAST nucleotide searches canbe performed with the NBLAST nucleotide program parameters set, e.g.,for score=100, wordlength=12 to obtain nucleotide sequences homologousto a nucleic acid molecules described herein. BLAST protein searches canbe performed with the XBLAST program parameters set, e.g., to score 50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecule described herein. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul S F etal., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated byreference in its entirety. Alternatively, PSI BLAST can be used toperform an iterated search which detects distant relationships betweenmolecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blastprograms, the default parameters of the respective programs (e.g., ofXBLAST and NBLAST) can be used (see, e.g., National Center forBiotechnology Information (NCBI) on the worldwide web,ncbi.nlm.nih.gov). Another specific, non-limiting example of amathematical algorithm utilized for the comparison of sequences is thealgorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is hereinincorporated by reference in its entirety. Such an algorithm isincorporated in the ALIGN program (version 2.0) which is part of the GCGsequence alignment software package. When utilizing the ALIGN programfor comparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used.

The percent identity between two sequences can be determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

6.2 Anti-CTLA-4 Antibodies

In one aspect the instant disclosure provides antibodies thatspecifically bind to CTLA-4 (e.g., human CTLA-4) and antagonize CTLA-4function. The amino acid sequences of exemplary antibodies are set forthin Tables 1-4 herein.

TABLE 1 Amino acid sequences of exemplary anti-CTLA-4 antibodies.* SEQID NO: Description Amino acid sequence  1 AGEN1884 VHEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM NWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGL MGPFDIWGQGTMVTVSS  2 AGEN1884_M102F VHEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM NWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF GPFDIWGQGTMVTVSS  3 AGEN1884_M113L VHEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM NWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGL MGPFDIWGQGTLVTVSS  4 AGEN1884_D62E VHEVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGL MGPFDIWGQGTMVTVSS  5AGEN1884_M102F_M113L  EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM VHNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLFGPFDIWGQGTLVTVSS  6 AGEN1884_D62E_ EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMM102F VH NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF GPFDIWGQGTMVTVSS  7AGEN1884_D62E_M113L  EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM VHNWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLMGPFDIWGQGTLVTVSS  8 AGEN1884_D62E_ EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMM102F_M113L VH NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF GPFDIWGQGTLVTVSS  9 AGEN1884 VLEIVLTQSPGTLSLSPGERATLSCRAQSVSRYLGW YQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTDFTLTITRLEPEDFAVYYCQQYGSSPWTFGQGTKVE IK 10 CDRH1 SYSMN 11 CDRH2SISSSSSYIYYADSVKG 12 CDRH2 SISSSSSYIYYAESVKG 13 CDRH3 VGLMGPFDI 14 CDRH3VGLFGPFDI 15 CDRL1 RASQSVSRYLG 16 CDRL2 GASTRAT 17 CDRL3 QQYGSSPWT 18CDRH2 consensus SISSSSSYIYYAXSVKG,wherein: sequence X is E or D 19CDRH3 consensus VGLXGPFDI, wherein: sequence X is For M 20VH consensus sequence EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYAX₁SVKGRF TISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLX₂GPFDIWGQGTX₃VTVSSS, wherein: X₁ is E or D, X₂ is F or M, andX₃ is L or M. 23 AGEN1884.H3 (IgG₁) EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMheavy chain NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF GPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG 24 AGEN1884.H3 (IgG₁EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM S239D/I332E) heavyNWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF chainTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF GPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPD VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 25 AGEN1884.H3 (IgG1EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM S239D/A330L/1332E)NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF heavy chainTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF GPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPD VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG 26 AGEN1884.H3 (IgG₁EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM L235V/F243L/R292P/NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF Y300L/P396L) heavyTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF chainGPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKRVEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSK LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 47 AGEN1884.H3 (IgG₁ EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMN297A) heavy chain NWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF GPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG 48 AGEN1884.H3 (IgG₁EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSM S267E/L328F) heavyNWVRQAPGKGLEWVSSISSSSSYIYYAESVKGRF chainTISRDNAKNSLYLQMNSLRAEDTAVYYCARVGLF GPFDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSV FLFPPKPKDTLMISRTPEVTCVVVDVEHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKAFPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDI AVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PG 27 AGEN1884.H3 lightEIVLTQSPGTLSLSPGERATLSCRASQSVSRYLGW chainYQQKPGQAPRLLIYGASTRATGIPDRFSGSGSGTD FTLTITRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYP REAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC 28 IgG₁ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG 29IgG₁ S239D/I332E ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 30 IgG₁ ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPES239D/A330L/I332E PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRE EMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPG 31 IgG₁ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE L235V/F243L/R292P/PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV Y300L/P396LTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC DKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPG 32Light chain constant RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR regionEAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC *CDRs are defined according to the Kabat numbering system.

TABLE 2Heavy chain CDR amino acid sequences of exemplary anti-CTLA-4 antibodies.*CDRH1 CDRH2 CDRH3 VH (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)AGEN1884 VH (1) SYSMN (10) SISSSSSYIYYADSV VGLMGPFDI (13) KG (11)AGEN1884_M102F VH (2) SYSMN (10) SISSSSSYIYYADSV VGLFGPFDI (14) KG (11)AGEN1884_M113L VH (3) SYSMN (10) SISSSSSYIYYADSV VGLMGPFDI (13) KG (11)AGEN1884_D62E VH (4) SYSMN (10) SISSSSSYIYYAESV VGLMGPFDI (13) KG (12)AGEN1884_M102F_M113L SYSMN (10) SISSSSSYIYYADSV VGLFGPFDI (14) VH (5)KG (11) AGEN1884_D62E_M102F SYSMN (10) SISSSSSYIYYAESV VGLFGPFDI (14)VH (6) KG (12) AGEN1884_D62E_M113L SYSMN (10) SISSSSSYIYYAESVVGLMGPFDI (13) VH (7) KG (12) AGEN1884_D62E_M102F SYSMN (10)SISSSSSYIYYAESV VGLFGPFDI (14) M113L VH (8) KG (12) *Defined accordingto the Kabat numbering system.

TABLE 3Light chain CDR amino acid sequences of exemplary anti-CTLA-4 antibodies.*VL CDRL1 CDRL2 CDRL3 (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:)AGEN1884 VL (9) RASQSVSRYLG (15) GASTRAT (16) QQYGSSPWT (17) *Definedaccording to the Kabat numbering system.

TABLE 4 Exemplary anti-CTLA-4 antibodies. SEQ SEQ Antibody Heavy chainvariable region ID NO: Light chain variable region ID NO: AGEN1884AGEN1884 VH 1 AGEN1884 VL 9 AGEN1884.H1.1 AGEN1884_M102F VH 2 AGEN1884VL 9 AGEN1884.H1.2 AGEN1884_M113L VH 3 AGEN1884 VL 9 AGEN1884.H1.3AGEN1884_D62E VH 4 AGEN1884 VL 9 AGEN1884.H2.1 AGEN1884_M102F_M113L VH 5AGEN1884 VL 9 AGEN1884.H2.2 AGEN1884_D62E_M102F VH 6 AGEN1884 VL 9AGEN1884.H2.3 AGEN1884_D62E_M113L VH 7 AGEN1884 VL 9 AGEN1884.H3AGEN1884_D62E_M102F_M113L VH 8 AGEN1884 VL 9

TABLE 5 Closest germline genes. SEQ ID Closest NO: germline geneAmino acid sequence 21 IGHV3-21*01 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVK GRFTISRDNAKNSLYLQMNSLRAEDTAVYYCA R 22IGKV3-20*01 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYCQQYGSSP

TABLE 6 Exemplary sequences of CTLA-4 and family members. SEQ ID NO:Description Amino acid Sequence 33 Human CTLA-4 immatureMACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFC protein (P16410)KAMHVAQPAVVLASSRGIASFVCEYASPGKATEV RVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYP PPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPP TEPECEKQFQPYFIPIN 34 CTLA-4 epitopeYLGI 35 CTLA-4 epitope YPPPYYLGI 36 CTLA-4 epitope YLGIGNGTQI 37CTLA-4 epitope YPPPYYLGIGNGTQI 38 CTLA-4 epitope MYPPPYY 39CTLA-4 epitope QVT 40 MACFA CTLA-4 MACLGFQRHKARLNLATRTRPYTLLFSLLFIPVFS(G7PL88) KAMHVAQPAVVLANSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDS ICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYMGIGNGTQIYVIDPEPCPDSDFLLWILAAVS SGLFFYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN 41 Mouse CTLA-4 (P09793)MACLGLRRYKAQLQLPSRTWPFVALLTLLFIPVFS EAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEKNTVGFLDYPFCS GTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQIYVIDPEPCPDSDFLLWILVAVSLG LFFYSFLVSAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN 42 Rat CTLA-4 (Q62859)MACLGLQRYKTHLQLPSRTWPFGVLLSLLFIPIFSE AIQVTQPSVVLASSHGVASFPCEYASSHNTDEVRVTVLRQTNDQVTEVCATTFTVKNTLGFLDDPFCS GTFNESRVNLTIQGLRAADTGLYFCKVELMYPPPYFVGMGNGTQIYVIDPEPCPDSDFLLWILAAVSSG LFFYSFLVTAVSLNRTLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN 43 Human CD28 (P10747)MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDN AVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQN LYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLV TVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 44 Human ICOS (Q9Y6W8)MKSGLWYFFLFCLRIKVLTGEINGSANYEMFIFHN GGVQILCKYPDIVQQFKMQLLKGGQILCDLTKTKGSGNTVSIKSLKFCHSQLSNNSVSFFLYNLDHSHA NYYFCNLSIFDPPPFKVTLTGGYLHIYESQLCCQLKFWLPIGCAAFVVVCILGCILICWLTKKKYSSSVH DPNGEYMFMRAVNTAKKSRLTDVTL 45Human BTLA (Q7Z6A9) MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHV TWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSA SERPSKDEMASRPWLLYRLLPLGGLPLLITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQT EASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKE APTEYASICVRS 46 Human PD-1 (Q15116)MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPW NPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLP NGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV VGVVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPP VPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising a VH domain comprising one, two, or all three of theCDRs of a VH domain set forth in Table 1 herein. In certain embodiments,the antibody comprises the CDRH1 of one of VH domains set forth inTable 1. In certain embodiments, the antibody comprises the CDRH2 of oneof the VH domains set forth in Table 1. In certain embodiments, theantibody comprises the CDRH3 of one of the VH domains set forth in Table1.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising a VL domain comprising one, two, or all three of theCDRs of a VL domain disclosed in Table 1 herein. In certain embodiments,the antibody comprises the CDRL1 of one of VL domains set forth inTable 1. In certain embodiments, the antibody comprises the CDRL2 of oneof the VL domains set forth in Table 1. In certain embodiments, theantibody comprises the CDRL3 of one of the VL domains set forth in Table1.

In certain embodiments, the CDRs of an antibody can be determinedaccording to Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) andKabat et al., Sequences of protein of immunological interest (1991),each of which is herein incorporated by reference in its entirety.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the Chothia numbering scheme, which refers to the locationof immunoglobulin structural loops (see, e.g., Chothia C & Lesk A M,(1987), J Mol Biol 196: 901-917; Al-Lazikani B et al., (1997) J Mol Biol273: 927-948; Chothia C et al., (1992) J Mol Biol 227: 799-817;Tramontano A et al., (1990) J Mol Biol 215(1): 175-82; and U.S. Pat. No.7,709,226, all of which are herein incorporated by reference in theirentireties). Typically, when using the Kabat numbering convention, theChothia CDRH1 loop is present at heavy chain amino acids 26 to 32, 33,or 34, the Chothia CDRH2 loop is present at heavy chain amino acids 52to 56, and the Chothia CDRH3 loop is present at heavy chain amino acids95 to 102, while the Chothia CDRL1 loop is present at light chain aminoacids 24 to 34, the Chothia CDRL2 loop is present at light chain aminoacids 50 to 56, and the Chothia CDRL3 loop is present at light chainamino acids 89 to 97. The end of the Chothia CDRH1 loop when numberedusing the Kabat numbering convention varies between H32 and H34depending on the length of the loop (this is because the Kabat numberingscheme places the insertions at H35A and H35B; if neither 35A nor 35B ispresent, the loop ends at 32; if only 35A is present, the loop ends at33; if both 35A and 35B are present, the loop ends at 34).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising the Chothia VH CDRs of a VH disclosed in Table 1herein. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CTLA-4 (e.g., humanCTLA-4), the antibody comprising the Chothia VL CDRs of a VL disclosedin Table 1 herein. In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CTLA-4 (e.g.,human CTLA-4), the antibody comprising the Chothia VH CDRs and ChothiaVL CDRs of an antibody disclosed in Table 1 herein. In certainembodiments, antibodies that specifically bind to CTLA-4 (e.g., humanCTLA-4) comprise one or more CDRs, in which the Chothia and Kabat CDRshave the same amino acid sequence. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCTLA-4 (e.g., human CTLA-4) and comprises combinations of Kabat CDRs andChothia CDRs.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the IMGT numbering system as described in Lefranc M-P,(1999) The Immunologist 7: 132-136 and Lefranc M-P et al., (1999)Nucleic Acids Res 27: 209-212, each of which is herein incorporated byreference in its entirety.

In certain embodiments, the instant disclosure provides antibodies thatspecifically bind to CTLA-4 (e.g., human CTLA-4) and comprise CDRs of anantibody disclosed in Table 1 herein, as determined by the IMGTnumbering system, for example, as described in Lefranc M-P (1999) supraand Lefranc M-P et al., (1999) supra.

In certain embodiments, the CDRs of an antibody can be determinedaccording to the AbM numbering scheme, which refers to AbM hypervariableregions, which represent a compromise between the Kabat CDRs and Chothiastructural loops, and are used by Oxford Molecular's AbM antibodymodeling software (Oxford Molecular Group, Inc.), herein incorporated byreference in its entirety. In a particular embodiment, the instantdisclosure provides antibodies that specifically bind to CTLA-4 (e.g.,human CTLA-4) and comprise CDRs of an antibody disclosed in Table 1herein as determined by the AbM numbering scheme.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), whereinthe antibody comprises a heavy chain variable region comprising theCDRH1, CDRH2, and CDRH3 region amino acid sequences of a VH domain setforth in SEQ ID NO: 2, 4, 5, 6, 7, or 8, and a light chain variableregion comprising the CDRL1, CDRL2, and CDRL3 region amino acidsequences of a VL domain set forth in SEQ ID NO: 9, wherein each CDR isdefined in accordance with the MacCallum definition, the Kabatdefinition, the Chothia definition, the combination of the Kabatdefinition and the Chothia definition, the IMGT numbering system, or theAbM definition of CDR.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising:

(a) a CDRH1 comprises the amino acid sequence of SYSMN (SEQ ID NO: 10);and/or(b) a CDRH2 comprises the amino acid sequence of SISSSSSYIYYAXSVKG (SEQID NO: 18), wherein X is E or D; and/or(c) a CDRH3 comprises the amino acid sequence of VGLXGPFDI (SEQ ID NO:19), wherein X is F or M; and/or(d) CDRL1 comprises the amino acid sequence of RASQSVSRYLG (SEQ ID NO:15); and/or(e) CDRL2 comprises the amino acid sequence of GASTRAT (SEQ ID NO: 16);and/or(f) CDRL3 comprises the amino acid sequence of QQYGSSPWT (SEQ ID NO:17), and wherein the CDRH1, CDRH2, and CDRH3 sequences of the antibodyare not SEQ ID NOs: 10, 11, and 13, respectively.

In certain embodiments, CDRH2 comprises the amino acid sequence of SEQID NO: 11. In certain embodiments, CDRH2 comprises the amino acidsequence of SEQ ID NO: 12. In certain embodiments, CDRH3 comprises theamino acid sequence of SEQ ID NO: 13. In certain embodiments, CDRH3comprises the amino acid sequence of SEQ ID NO: 14.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), whereinthe antibody comprises a VH domain comprising the CDRH1, CDRH2, andCDRH3 amino acid sequences set forth in SEQ ID NOs: 10, 11, and 14; 10,12, and 13; or 10, 12, and 14, respectively. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CTLA-4 (e.g., human CTLA-4), wherein the antibody comprises a VHdomain comprising the CDRH1, CDRH2, and CDRH3 amino acid sequences setforth in SEQ ID NOs: 10, 12, and 14, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), whereinthe antibody comprises a heavy chain variable region comprising CDRH1,CDRH2, and CDRH3 regions, and a light chain variable region comprisingCDRL1, CDRL2, and CDRL3 regions, wherein the CDRH1, CDRH2, CDRH3, CDRL1,CDRL2, and CDRL3 regions comprise the amino acid sequences set forth inSEQ ID NOs: 10, 11, 14, 15, 16, and 17; 10, 12, 13, 15, 16, and 17; or10, 12, 14, 15, 16, and 17, respectively. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CTLA-4 (e.g., human CTLA-4), wherein the antibody comprises a heavychain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and alight chain variable region comprising CDRL1, CDRL2, and CDRL3 regions,wherein the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 regionscomprise the amino acid sequences set forth in SEQ ID NOs: 10, 12, 14,15, 16, and 17, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 20. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable regioncomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, 99%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth inSEQ ID NO: 2, 4, 5, 6, 7, or 8. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable regioncomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, 99%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth inSEQ ID NO: 3. In certain embodiments, the antibody comprises a heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 2, 4, 5, 6, 7, or 8. In certain embodiments, the antibody comprisesa heavy chain variable region having the amino acid sequence set forthin SEQ ID NO: 2. In certain embodiments, the antibody comprises a heavychain variable region having the amino acid sequence set forth in SEQ IDNO: 3. In certain embodiments, the antibody comprises a heavy chainvariable region having the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the antibody comprises a heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO: 5. Incertain embodiments, the antibody comprises a heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO: 6. Incertain embodiments, the antibody comprises a heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO: 7. Incertain embodiments, the antibody comprises a heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO: 8.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4),comprising a light chain variable region comprising an amino acidsequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100% (e.g.,at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%)identical to the amino acid sequence set forth in SEQ ID NO: 9. Incertain embodiments, the antibody comprises a light chain variableregion having the amino acid sequence set forth in SEQ ID NO: 9.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4),comprising a heavy chain variable region comprising an amino acidsequence of SEQ ID NO: 20, and a light chain variable region comprisingan amino acid sequence of SEQ ID NO: 9. In certain embodiments, theinstant disclosure provides an isolated antibody that specifically bindsto CTLA-4 (e.g., human CTLA-4), comprising a heavy chain variable regioncomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, 99%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth inSEQ ID NO: 2, 4, 5, 6, 7, or 8, and a light chain variable regioncomprising an amino acid sequence that is at least 75%, 80%, 85%, 90%,95%, 99%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth inSEQ ID NO: 9. In certain embodiments, the instant disclosure provides anisolated antibody that specifically binds to CTLA-4 (e.g., humanCTLA-4), comprising a heavy chain variable region comprising an aminoacid sequence that is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%(e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or99%) identical to the amino acid sequence set forth in SEQ ID NO: 3, anda light chain variable region comprising an amino acid sequence that isat least 75%, 80%, 85%, 90%, 95%, 99%, or 100% (e.g., at least 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to theamino acid sequence set forth in SEQ ID NO: 9. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:2 and 9; 4 and 9; 5 and 9; 6 and 9; 7 and 9; or 8 and 9, respectively.In certain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 2 and 9, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:3 and 9, respectively. In certain embodiments, the antibody comprises aheavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 4 and 9, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 5 and 9, respectively. In certain embodiments,the antibody comprises a heavy chain variable region and light chainvariable region having the amino acid sequences set forth in SEQ ID NO:6 and 9, respectively. In certain embodiments, the antibody comprises aheavy chain variable region and light chain variable region having theamino acid sequences set forth in SEQ ID NO: 7 and 9, respectively. Incertain embodiments, the antibody comprises a heavy chain variableregion and light chain variable region having the amino acid sequencesset forth in SEQ ID NO: 8 and 9, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4),comprising a heavy chain variable region having an amino acid sequencederived from a human IGHV3-21 germline sequence (e.g., IGHV3-21*01,e.g., having the amino acid sequence of SEQ ID NO: 21). One or moreregions selected from framework 1, framework 2, framework 3, CDRH1, andCDRH2 (e.g., two, three, four or five of these regions) can be derivedfrom a human IGHV3-21 germline sequence (e.g., IGHV3-21*01, e.g., havingthe amino acid sequence of SEQ ID NO: 21). In one embodiment, framework1, framework 2, framework 3, CDRH1, and CDRH2 are all derived from ahuman IGHV3-21 germline sequence (e.g., IGHV3-21*01, e.g., having theamino acid sequence of SEQ ID NO: 21).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4),comprising a light chain variable region having an amino acid sequencederived from a human germline sequence selected from the groupconsisting of IGKV3-20 (e.g., IGKV3-20*01, e.g., having the amino acidsequence of SEQ ID NO: 22). One or more regions selected from framework1, framework 2, framework 3, CDRL1, and CDRL2 (e.g., two, three, four orfive of these regions) can be derived from a human germline sequenceselected from the group consisting of IGKV3-20 (e.g., IGKV3-20*01, e.g.,having the amino acid sequence of SEQ ID NO: 22). In one embodiment,framework 1, framework 2, framework 3, CDRL1, and CDRL2 are all derivedfrom a human germline sequence selected from the group consisting ofIGKV3-20 (e.g., IGKV3-20*01, e.g., having the amino acid sequence of SEQID NO: 22).

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4),comprising a heavy chain variable region having an amino acid sequencederived from a human IGHV3-21 germline sequence (e.g., IGHV3-21*01,e.g., having the amino acid sequence of SEQ ID NO: 21), and a lightchain variable region having an amino acid sequence derived from a humangermline sequence selected from the group consisting of IGKV3-20 (e.g.,IGKV3-20*01, e.g., having the amino acid sequence of SEQ ID NO: 22).

In certain embodiments, the instant disclosure provides an isolatedantibody that cross-competes for binding to CTLA-4 (e.g., human CTLA-4)with an antibody comprising the heavy and light chain variable regionamino acid sequences set forth in SEQ ID NOs: 2 and 9; 4 and 9; 5 and 9;6 and 9; 7 and 9; or 8 and 9, respectively. In certain embodiments, theinstant disclosure provides an isolated antibody that cross-competes forbinding to CTLA-4 (e.g., human CTLA-4) with an antibody comprising theheavy and light chain variable region amino acid sequences set forth inSEQ ID NOs: 3 and 9, respectively.

In certain embodiments, the instant disclosure provides an isolatedantibody that binds to the same or an overlapping epitope of CTLA-4(e.g., an epitope of human CTLA-4) as an antibody described herein,e.g., an antibody comprising the heavy and light chain variable regionamino acid sequences set forth in SEQ ID NOs: 2 and 9; 4 and 9; 5 and 9;6 and 9; 7 and 9; or 8 and 9, respectively. In certain embodiments, theinstant disclosure provides an isolated antibody that binds to the sameor an overlapping epitope of CTLA-4 (e.g., an epitope of human CTLA-4)as an antibody described herein, e.g., an antibody comprising the heavyand light chain variable region amino acid sequences set forth in SEQ IDNOs: 3 and 9, respectively. In certain embodiments, the epitope of anantibody can be determined by, e.g., NMR spectroscopy, surface plasmonresonance (BIAcore®), X-ray diffraction crystallography studies, ELISAassays, hydrogen/deuterium exchange coupled with mass spectrometry(e.g., liquid chromatography electrospray mass spectrometry),array-based oligo-peptide scanning assays, and/or mutagenesis mapping(e.g., site-directed mutagenesis mapping). For X-ray crystallography,crystallization may be accomplished using any of the known methods inthe art (e.g., Giegé R et al., (1994) Acta Crystallogr D BiolCrystallogr 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189:1-23; Chayen N E (1997) Structure 5: 1269-1274; McPherson A (1976) JBiol Chem 251: 6300-6303, all of which are herein incorporated byreference in their entireties). Antibody:antigen crystals may be studiedusing well known X-ray diffraction techniques and may be refined usingcomputer software such as X-PLOR (Yale University, 1992, distributed byMolecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114& 115, eds Wyckoff H W et al.; U.S. Patent Application No.2004/0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D BiolCrystallogr 49(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter C W; Roversi P et al., (2000) Acta Crystallogr D BiolCrystallogr 56(Pt 10): 1316-1323, all of which are herein incorporatedby reference in their entireties). Mutagenesis mapping studies may beaccomplished using any method known to one of skill in the art. See,e.g., Champe M et al., (1995) supra and Cunningham B C & Wells J A(1989) supra for a description of mutagenesis techniques, includingalanine scanning mutagenesis techniques. In a specific embodiment, theepitope of an antibody is determined using alanine scanning mutagenesisstudies. In addition, antibodies that recognize and bind to the same oroverlapping epitopes of CTLA-4 (e.g., human CTLA-4) can be identifiedusing routine techniques such as an immunoassay, for example, by showingthe ability of one antibody to block the binding of another antibody toa target antigen, i.e., a competitive binding assay. Competition bindingassays also can be used to determine whether two antibodies have similarbinding specificity for an epitope. Competitive binding can bedetermined in an assay in which the immunoglobulin under test inhibitsspecific binding of a reference antibody to a common antigen, such asCTLA-4 (e.g., human CTLA-4). Numerous types of competitive bindingassays are known, for example: solid phase direct or indirectradioimmunoassay (RIA), solid phase direct or indirect enzymeimmunoassay (EIA), sandwich competition assay (see Stahli C et al.,(1983) Methods Enzymol 9: 242-253); solid phase direct biotin-avidin EIA(see Kirkland T N et al., (1986) J Immunol 137: 3614-9); solid phasedirect labeled assay, solid phase direct labeled sandwich assay (seeHarlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold SpringHarbor Press); solid phase direct label RIA using I-125 label (see MorelG A et al., (1988) Mol Immunol 25(1): 7-15); solid phase directbiotin-avidin EIA (see Cheung R C et al., (1990) Virology 176: 546-52);and direct labeled RIA (see Moldenhauer G et al., (1990) Scand J Immunol32: 77-82), all of which are herein incorporated by reference in theirentireties. Typically, such an assay involves the use of purifiedantigen (e.g., CTLA-4 such as human CTLA-4) bound to a solid surface orcells bearing either of these, an unlabeled test immunoglobulin and alabeled reference immunoglobulin. Competitive inhibition can be measuredby determining the amount of label bound to the solid surface or cellsin the presence of the test immunoglobulin. Usually the testimmunoglobulin is present in excess. Usually, when a competing antibodyis present in excess, it will inhibit specific binding of a referenceantibody to a common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%,70-75% or more. A competition binding assay can be configured in a largenumber of different formats using either labeled antigen or labeledantibody. In a common version of this assay, the antigen is immobilizedon a 96-well plate. The ability of unlabeled antibodies to block thebinding of labeled antibodies to the antigen is then measured usingradioactive or enzyme labels. For further details see, for example,Wagener C et al., (1983) J Immunol 130: 2308-2315; Wagener C et al.,(1984) J Immunol Methods 68: 269-274; Kuroki M et al., (1990) Cancer Res50: 4872-4879; Kuroki M et al., (1992) Immunol Invest 21: 523-538;Kuroki M et al., (1992) Hybridoma 11: 391-407 and Antibodies: ALaboratory Manual, Ed Harlow E & Lane D editors supra, pp. 386-389, allof which are herein incorporated by reference in their entireties.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 23, 24, 25, or 26. In certain embodiments, theantibody comprises a heavy chain comprising the amino acid sequence setforth in SEQ ID NO: 23. In certain embodiments, the antibody comprises aheavy chain comprising the amino acid sequence set forth in SEQ ID NO:24. In certain embodiments, the antibody comprises a heavy chaincomprising the amino acid sequence set forth in SEQ ID NO: 25. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence set forth in SEQ ID NO: 26.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising a light chain comprising the amino acid sequence setforth in SEQ ID NO: 27.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising a heavy chain comprising the amino acid sequence ofSEQ ID NO: 23 and a light chain comprising the amino acid sequence ofSEQ ID NO: 27. In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to CTLA-4 (e.g., humanCTLA-4), the antibody comprising a heavy chain comprising the amino acidsequence of SEQ ID NO: 24 and a light chain comprising the amino acidsequence of SEQ ID NO: 27. In certain embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCTLA-4 (e.g., human CTLA-4), the antibody comprising a heavy chaincomprising the amino acid sequence of SEQ ID NO: 25 and a light chaincomprising the amino acid sequence of SEQ ID NO: 27. In certainembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CTLA-4 (e.g., human CTLA-4), the antibodycomprising a heavy chain comprising the amino acid sequence of SEQ IDNO: 26 and a light chain comprising the amino acid sequence of SEQ IDNO: 27.

Any Ig constant region can be used in the antibodies described herein.In certain embodiments, the Ig region is a human IgG, IgE, IgM, IgD,IgA, or IgY immunoglobulin molecule, any class (e.g., IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂), or any subclass (e.g., IgG_(2a) and IgG_(2b)) ofimmunoglobulin molecule.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising a heavy chain constant region comprising the aminoacid sequence of SEQ ID NO: 28, 29, 30, or 31. In certain embodiments,the instant disclosure provides an isolated antibody that specificallybinds to CTLA-4 (e.g., human CTLA-4), the antibody comprising a lightchain constant region comprising the amino acid sequence of SEQ ID NO:32.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising a heavy chain constant region, e.g., an IgG₁constant region, or fragment thereof comprising a mutation selected fromthe group consisting of: S239D, I332E, and a combination thereof,numbered according to the EU numbering system. In certain embodiments,the antibody comprises an IgG₁ heavy chain constant region comprisingS239D and I332E mutations, numbered according to the EU numberingsystem. In certain embodiments, the antibody comprises a heavy chainconstant region comprising the amino acid sequence of SEQ ID NO: 29.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising a heavy chain constant region, e.g., an IgG₁constant region, or fragment thereof comprising a mutation selected fromthe group consisting of: S239D, A330L, I332E, and combinations thereof,numbered according to the EU numbering system. In certain embodiments,the antibody comprises an IgG₁ heavy chain constant region comprisingS239D, A330L, and I332E mutations, numbered according to the EUnumbering system. In certain embodiments, the antibody comprises a heavychain constant region comprising the amino acid sequence of SEQ ID NO:30.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4), theantibody comprising a heavy chain constant region, e.g., an IgG₁constant region, or fragment thereof comprising a mutation selected fromthe group consisting of: L235V, F243L, R292P, Y300L, P396L, andcombinations thereof, numbered according to the EU numbering system. Incertain embodiments, the antibody comprises an IgG₁ heavy chain constantregion comprising L235V, F243L, R292P, Y300L, and P396L mutations,numbered according to the EU numbering system. In certain embodiments,the antibody comprises a heavy chain constant region comprising theamino acid sequence of SEQ ID NO: 31.

In certain embodiments, the IgG regions of the antibodies describedherein have an increased affinity for FcγRIIIA, e.g., as compared withan antibody with a wild-type Fc region, e.g., an IgG₁ Fc. Sequencealterations that result in increased affinity for FcγRIIIA are known inthe art, for example, in Kellner et al., Methods 65: 105-113 (2014),Lazar et al., Proc Natl Acad Sci 103: 4005-4010 (2006), Shields et al.,J Biol Chem. 276(9):6591-6604 (2001), each of which is hereinincorporated by reference in its entirety. In certain embodiments, theantibody comprises a heavy chain constant region, e.g., an IgG₁ constantregion, or fragment thereof comprising a mutation selected from thegroup consisting of: G236A, S239D, F243L, T256A, K290A, R292P, S298A,Y300L, V305I, A330L, I332E, E333A, K334A, A339T, and P396L, andcombinations thereof, numbered according to the EU numbering system. Incertain embodiments, the antibody comprises a heavy chain constantregion, e.g., an IgG1 constant region, or fragment thereof comprisingS239D, numbered according to the EU numbering system. In certainembodiments, the antibody comprises a heavy chain constant region, e.g.,an IgG1 constant region, or fragment thereof comprising T256A, numberedaccording to the EU numbering system. In certain embodiments, theantibody comprises a heavy chain constant region, e.g., an IgG1 constantregion, or fragment thereof comprising K290A, numbered according to theEU numbering system. In certain embodiments, the antibody comprises aheavy chain constant region, e.g., an IgG1 constant region, or fragmentthereof comprising S298A, numbered according to the EU numbering system.In certain embodiments, the antibody comprises a heavy chain constantregion, e.g., an IgG1 constant region, or fragment thereof comprisingI332E, numbered according to the EU numbering system. In certainembodiments, the antibody comprises a heavy chain constant region, e.g.,an IgG1 constant region, or fragment thereof comprising E333A, numberedaccording to the EU numbering system. In certain embodiments, theantibody comprises a heavy chain constant region, e.g., an IgG1 constantregion, or fragment thereof comprising K334A, numbered according to theEU numbering system. In certain embodiments, the antibody comprises aheavy chain constant region, e.g., an IgG1 constant region, or fragmentthereof comprising A339T, numbered according to the EU numbering system.In certain embodiments, the antibody comprises a heavy chain constantregion, e.g., an IgG1 constant region, or fragment thereof comprisingS239D and I332E, numbered according to the EU numbering system. Incertain embodiments, the antibody comprises a heavy chain constantregion, e.g., an IgG1 constant region, or fragment thereof comprisingS239D, A330L, and I332E, numbered according to the EU numbering system.In certain embodiments, the antibody comprises a heavy chain constantregion, e.g., an IgG1 constant region, or fragment thereof comprisingS298A, E333A, and K334A, numbered according to the EU numbering system.In certain embodiments, the antibody comprises a heavy chain constantregion, e.g., an IgG1 constant region, or fragment thereof comprisingG236A, S239D, and I332E, numbered according to the EU numbering system.In certain embodiments, the antibody comprises a heavy chain constantregion, e.g., an IgG1 constant region, or fragment thereof comprisingF243L, R292P, Y300L, V305I, and P396L, numbered according to the EUnumbering system.

In certain embodiments, the antibodies described herein exhibitantibody-dependent cellular cytotoxicity (ADCC) activity. In certainembodiments, the antibodies described herein initiate natural killercell mediated cell depletion. In certain embodiments, the antibodiesdescribed herein are used for treating tumor infiltrated with naturalkiller cells. In certain embodiments, the antibodies described hereinexhibit antibody-dependent cellular phagocytosis (ADCP) activity. Incertain embodiments, the antibodies described herein initiate macrophagemediated cell depletion. In certain embodiments, the antibodiesdescribed herein are used for treating tumor infiltrated withmacrophages. In certain embodiments, the antibodies described hereinselectively deplete intratumoral regulatory T cells.

In certain embodiments, an antibody described herein is an activatableantibody that in an activated state binds human CTLA-4 protein. Incertain embodiments, the activatable antibody comprises a masking moietythat inhibits the binding of the antibody in an uncleaved state to humanCTLA-4 protein, and at least one cleavable moiety coupled to theantibody, e.g., wherein the cleavable moiety is a polypeptide thatfunctions as a substrate for a protease that is enriched in the tumormicroenvironment. Exemplary activatable antibodies are described, e.g.,in U.S. Pat. Nos. 8,513,390 and 8,518,404, and U.S. Patent ApplicationPublication Nos. US 2014/0255313, US 2014/0010810, US 2014/0023664,which are incorporated herein by reference. In certain embodiments, theactivatable antibody comprises a human IgG heavy chain constant regionthat is a variant of a wild type human IgG heavy chain constant region,wherein the variant human IgG heavy chain constant region binds to humanFcγRIIIA with higher affinity than the wild type human IgG heavy chainconstant region binds to human FcγRIIIA

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system, to alter one ormore functional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding and/or antigen-dependentcellular cytotoxicity.

In certain embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the hinge region of the Fc region(CH1 domain) such that the number of cysteine residues in the hingeregion are altered (e.g., increased or decreased) as described in, e.g.,U.S. Pat. No. 5,677,425, herein incorporated by reference in itsentirety. The number of cysteine residues in the hinge region of the CH1domain may be altered to, e.g., facilitate assembly of the light andheavy chains, or to alter (e.g., increase or decrease) the stability ofthe antibody.

In a specific embodiment, one, two, or more amino acid mutations (e.g.,substitutions, insertions or deletions) are introduced into an IgGconstant domain, or FcRn-binding fragment thereof (preferably an Fc orhinge-Fc domain fragment) to alter (e.g., decrease or increase)half-life of the antibody in vivo. See, e.g., International PublicationNos. WO 02/060919; WO 98/23289; and WO 97/34631; and U.S. Pat. Nos.5,869,046, 6,121,022, 6,277,375 and 6,165,745, all of which are hereinincorporated by reference in their entireties, for examples of mutationsthat will alter (e.g., decrease or increase) the half-life of anantibody in vivo. In some embodiments, one, two or more amino acidmutations (e.g., substitutions, insertions, or deletions) are introducedinto an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to decrease the half-lifeof the antibody in vivo. In other embodiments, one, two or more aminoacid mutations (e.g., substitutions, insertions or deletions) areintroduced into an IgG constant domain, or FcRn-binding fragment thereof(preferably an Fc or hinge-Fc domain fragment) to increase the half-lifeof the antibody in vivo. In a specific embodiment, the antibodies mayhave one or more amino acid mutations (e.g., substitutions) in thesecond constant (CH2) domain (residues 231-340 of human IgG₁) and/or thethird constant (CH3) domain (residues 341-447 of human IgG₁), numberedaccording to the EU numbering system. In a specific embodiment, theconstant region of the IgG₁ of an antibody described herein comprises amethionine (M) to tyrosine (Y) substitution in position 252, a serine(S) to threonine (T) substitution in position 254, and a threonine (T)to glutamic acid (E) substitution in position 256, numbered according tothe EU numbering system. See U.S. Pat. No. 7,658,921, which is hereinincorporated by reference in its entirety. This type of mutant IgG,referred to as “YTE mutant” has been shown to display fourfold increasedhalf-life as compared to wild-type versions of the same antibody (seeDall'Acqua W F et al., (2006) J Biol Chem 281: 23514-24, which is hereinincorporated by reference in its entirety). In certain embodiments, anantibody comprises an IgG constant domain comprising one, two, three ormore amino acid substitutions of amino acid residues at positions251-257, 285-290, 308-314, 385-389, and 428-436, numbered according tothe EU numbering system.

In some embodiments, one, two, or more mutations (e.g., amino acidsubstitutions) are introduced into the Fc region of an antibodydescribed herein (e.g., CH2 domain (residues 231-340 of human IgG₁)and/or CH3 domain (residues 341-447 of human IgG₁) and/or the hingeregion, numbered according to the EU numbering system, to increase ordecrease the affinity of the antibody for an Fc receptor (e.g., anactivated Fc receptor) on the surface of an effector cell. Mutations inthe Fc region of an antibody that decrease or increase the affinity ofan antibody for an Fc receptor and techniques for introducing suchmutations into the Fc receptor or fragment thereof are known to one ofskill in the art. Examples of mutations in the Fc receptor of anantibody that can be made to alter the affinity of the antibody for anFc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos.WO 02/060919; WO 98/23289; and WO 97/34631, all of which are hereinincorporated by reference in their entireties.

In a further embodiment, one, two, or more amino acid substitutions areintroduced into an IgG constant domain Fc region to alter the effectorfunction(s) of the antibody. For example, one or more amino acidsselected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and322, numbered according to the EU numbering system, can be replaced witha different amino acid residue such that the antibody has an alteredaffinity for an effector ligand but retains the antigen-binding abilityof the parent antibody. The effector ligand to which affinity is alteredcan be, for example, an Fc receptor or the C1 component of complement.This approach is described in further detail in U.S. Pat. Nos. 5,624,821and 5,648,260, each of which is herein incorporated by reference in itsentirety. In some embodiments, the deletion or inactivation (throughpoint mutations or other means) of a constant region domain may reduceFc receptor binding of the circulating antibody thereby increasing tumorlocalization. See, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886, each ofwhich is herein incorporated by reference in its entirety, for adescription of mutations that delete or inactivate the constant domainand thereby increase tumor localization. In certain embodiments, one ormore amino acid substitutions may be introduced into the Fc region of anantibody described herein to remove potential glycosylation sites on Fcregion, which may reduce Fc receptor binding (see, e.g., Shields R L etal., (2001) J Biol Chem 276: 6591-604, which is herein incorporated byreference in its entirety). In various embodiments, one or more of thefollowing mutations in the constant region of an antibody describedherein may be made: an N297A substitution; an N297Q substitution; aL235A substitution and a L237A substitution; a L234A substitution and aL235A substitution; a E233P substitution; a L234V substitution; a L235Asubstitution; a C236 deletion; a P238A substitution; a D265Asubstitution; a A327Q substitution; or a P329A substitution, numberedaccording to the EU numbering system. In certain embodiments, a mutationselected from the group consisting of D265A, P329A, and a combinationthereof, numbered according to the EU numbering system, may be made inthe constant region of an antibody described herein.

In a specific embodiment, an antibody described herein comprises theconstant domain of an IgG₁ with an N297Q or N297A amino acidsubstitution, numbered according to the EU numbering system. In oneembodiment, an antibody described herein comprises the constant domainof an IgG₁ with a mutation selected from the group consisting of D265A,P329A, and a combination thereof, numbered according to the EU numberingsystem. In another embodiment, an antibody described herein comprisesthe constant domain of an IgG₁ with a mutation selected from the groupconsisting of L234A, L235A, and a combination thereof, numberedaccording to the EU numbering system. In certain embodiments, amino acidresidues in the constant region of an antibody described herein in thepositions corresponding to positions L234, L235, and D265 in a humanIgG₁ heavy chain, numbered according to the EU numbering system, are notL, L, and D, respectively. This approach is described in detail inInternational Publication No. WO 14/108483, which is herein incorporatedby reference in its entirety. In a particular embodiment, the aminoacids corresponding to positions L234, L235, and D265 in a human IgG₁heavy chain are F, E, and A; or A, A, and A, respectively, numberedaccording to the EU numbering system.

In certain embodiments, one or more amino acids selected from amino acidresidues 329, 331, and 322 in the constant region of an antibodydescribed herein, numbered according to the EU numbering system, can bereplaced with a different amino acid residue such that the antibody hasaltered C1q binding and/or reduced or abolished complement dependentcytotoxicity (CDC). This approach is described in further detail in U.S.Pat. No. 6,194,551 (Idusogie et al.), which is herein incorporated byreference in its entirety. In some embodiments, one or more amino acidresidues within amino acid positions 231 to 238 in the N-terminal regionof the CH2 domain of an antibody described herein are altered to therebyalter the ability of the antibody to fix complement, numbered accordingto the EU numbering system. This approach is described further inInternational Publication No. WO 94/29351, which is herein incorporatedby reference in its entirety. In certain embodiments, the Fc region ofan antibody described herein is modified to increase the ability of theantibody to mediate antibody dependent cellular cytotoxicity (ADCC)and/or to increase the affinity of the antibody for an Fcγ receptor bymutating one or more amino acids (e.g., introducing amino acidsubstitutions) at the following positions: 238, 239, 248, 249, 252, 254,255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285,286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309,312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335,337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419,430, 434, 435, 437, 438, or 439, numbered according to the EU numberingsystem. This approach is described further in International PublicationNo. WO 00/42072, which is herein incorporated by reference in itsentirety.

In certain embodiments, an antibody described herein comprises theconstant region of an IgG₄ antibody and the serine at amino acid residue228 of the heavy chain, numbered according to the EU numbering system,is substituted for proline.

In certain embodiments, any of the constant region mutations ormodifications described herein can be introduced into one or both heavychain constant regions of an antibody described herein having two heavychain constant regions.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4) andfunctions as an antagonist.

In certain embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4) anddecreases CTLA-4 (e.g., human CTLA-4) activity by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, 98%, or 99% as assessed by methods described herein and/orknown to one of skill in the art, relative to CTLA-4 (e.g., humanCTLA-4) activity without any antibody or with an unrelated antibody(e.g., an antibody that does not specifically bind to CTLA-4 (e.g.,human CTLA-4)). In certain embodiments, the instant disclosure providesan isolated antibody that specifically binds to CTLA-4 (e.g., humanCTLA-4) and decreases CTLA-4 (e.g., human CTLA-4) activity by at leastabout 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold,3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold, or 100 fold as assessed by methods described hereinand/or known to one of skill in the art, relative to CTLA-4 (e.g., humanCTLA-4) activity without any antibody or with an unrelated antibody(e.g., an antibody that does not specifically bind to CTLA-4 (e.g.,human CTLA-4)). Non-limiting examples of CTLA-4 (e.g., human CTLA-4)activity can include CTLA-4 (e.g., human CTLA-4) signaling, CTLA-4(e.g., human CTLA-4) binding to CTLA-4 (e.g., human CTLA-4) ligand(e.g., CD80 or CD86), and inhibition of cytokine production (e.g., IL-2,IFN-γ, or TNF-α). In certain embodiments, the instant disclosureprovides an isolated antibody that specifically binds to CTLA-4 (e.g.,human CTLA-4) and deactivates, reduces, or inhibits a CTLA-4 (e.g.,human CTLA-4) activity. In specific embodiments, a decrease in a CTLA-4(e.g., human CTLA-4) activity is assessed as described in the Examples,infra.

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4) andreduces CTLA-4 (e.g., human CTLA-4) binding to its ligand (e.g., CD80 orCD86) by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed bymethods known to one of skill in the art, relative to CTLA-4 (e.g.,human CTLA-4) binding to its ligand (e.g., CD80 or CD86) without anyantibody or with an unrelated antibody (e.g., an antibody that does notspecifically bind to CTLA-4 (e.g., human CTLA-4)). In specificembodiments, the instant disclosure provides an isolated antibody thatspecifically binds to CTLA-4 (e.g., human CTLA-4) and reduces CTLA-4(e.g., human CTLA-4) binding to its ligand (e.g., CD80 or CD86) by atleast about 1.2 fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9fold, 10 fold, 15 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70fold, 80 fold, 90 fold, or 100 fold, as assessed by methods known to oneof skill in the art, relative to CTLA-4 (e.g., human CTLA-4) binding toits ligand (e.g., CD80 or CD86) without any antibody or with anunrelated antibody (e.g., an antibody that does not specifically bind toCTLA-4 (e.g., human CTLA-4)).

In specific embodiments, the instant disclosure provides an isolatedantibody that specifically binds to CTLA-4 (e.g., human CTLA-4) andincreases cytokine production (e.g., IL-2, IFN-γ, or TNF-α) by at leastabout 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%, as assessed by methodsdescribed herein (see the Examples, infra) or known to one of skill inthe art, relative to cytokine production without any antibody or with anunrelated antibody (e.g., an antibody that does not specifically bind toCTLA-4 (e.g., human CTLA-4)). In specific embodiments, the instantdisclosure provides an isolated antibody that specifically binds toCTLA-4 (e.g., human CTLA-4) and increases cytokine production (e.g.,IL-2, IFN-γ, or TNF-α) by at least about 1.2 fold, 1.3 fold, 1.4 fold,1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold, 4 fold, 4.5 fold, 5 fold,6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15 fold, 20 fold, 30 fold, 40fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, or 100 fold, asassessed by methods described herein (see the Examples, infra) or knownto one of skill in the art, relative to cytokine production without anyantibody or with an unrelated antibody (e.g., an antibody that does notspecifically bind to CTLA-4 (e.g., human CTLA-4)).

In certain embodiments, human peripheral blood mononuclear cells (PBMCs)stimulated with Staphylococcus Enterotoxin A (SEA) in the presence of anantibody described herein, which specifically binds to CTLA-4 (e.g.,human CTLA-4), have increased IL-2 production by at least about 1.2fold, 1.3 fold, 1.4 fold, 1.5 fold, 2 fold, 2.5 fold, 3 fold, 3.5 fold,4 fold, 4.5 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 15fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90fold, or 100 fold relative to PBMCs only stimulated with SEA without anyantibody or with an unrelated antibody (e.g., an antibody that does notspecifically bind to CTLA-4 (e.g., human CTLA-4)), as assessed bymethods described herein (see the Examples, infra) or known to one ofskill in the art.

6.3 Pharmaceutical Compositions

Provided herein are compositions comprising an anti-CTLA-4 antibodydescribed herein having the desired degree of purity in aphysiologically acceptable carrier, excipient or stabilizer (Remington'sPharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.).Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In a specific embodiment, pharmaceutical compositions comprise ananti-CTLA-4 antibody described herein, and optionally one or moreadditional prophylactic or therapeutic agents, in a pharmaceuticallyacceptable carrier. In a specific embodiment, pharmaceuticalcompositions comprise an effective amount of an antibody orantigen-binding fragment thereof described herein, and optionally one ormore additional prophylactic or therapeutic agents, in apharmaceutically acceptable carrier. In some embodiments, the antibodyis the only active ingredient included in the pharmaceuticalcomposition. Pharmaceutical compositions described herein can be usefulin inhibiting, CTLA-4 activity and treating a condition, such as canceror an infectious disease.

In one aspect, provided herein is a pharmaceutical compositioncomprising an anti-CTLA-4 antibody of the invention and apharmaceutically acceptable carrier or excipient, for use as amedicament.

In one aspect, provided herein is a pharmaceutical compositioncomprising an anti-CTLA-4 antibody of the invention and apharmaceutically acceptable carrier or excipient, for use in a methodfor the treatment of cancer.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances. Examples ofaqueous vehicles include Sodium Chloride Injection, Ringers Injection,Isotonic Dextrose Injection, Sterile Water Injection, Dextrose andLactated Ringers Injection. Nonaqueous parenteral vehicles include fixedoils of vegetable origin, cottonseed oil, corn oil, sesame oil andpeanut oil. Antimicrobial agents in bacteriostatic or fungistaticconcentrations can be added to parenteral preparations packaged inmultiple-dose containers which include phenols or cresols, mercurials,benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acidesters, thimerosal, benzalkonium chloride and benzethonium chloride.Isotonic agents include sodium chloride and dextrose. Buffers includephosphate and citrate. Antioxidants include sodium bisulfate. Localanesthetics include procaine hydrochloride. Suspending and dispersingagents include sodium carboxymethylcelluose, hydroxypropylmethylcellulose and polyvinylpyrrolidone. Emulsifying agents includePolysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metalions includes EDTA. Pharmaceutical carriers also include ethyl alcohol,polyethylene glycol and propylene glycol for water miscible vehicles;and sodium hydroxide, hydrochloric acid, citric acid or lactic acid forpH adjustment.

A pharmaceutical composition may be formulated for any route ofadministration to a subject. Specific examples of routes ofadministration include intranasal, oral, pulmonary, transdermal,intradermal, and parenteral. Parenteral administration, characterized byeither subcutaneous, intramuscular or intravenous injection, is alsocontemplated herein. Injectables can be prepared in conventional forms,either as liquid solutions or suspensions, solid forms suitable forsolution or suspension in liquid prior to injection, or as emulsions.The injectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered can also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Preparations for parenteral administration of an antibody includesterile solutions ready for injection, sterile dry soluble products,such as lyophilized powders, ready to be combined with a solvent justprior to use, including hypodermic tablets, sterile suspensions readyfor injection, sterile dry insoluble products ready to be combined witha vehicle just prior to use and sterile emulsions. The solutions may beeither aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Topical mixtures comprising an antibody are prepared as described forthe local and systemic administration. The resulting mixture can be asolution, suspension, emulsions or the like and can be formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

An anti-CTLA-4 antibody described herein can be formulated as an aerosolfor topical application, such as by inhalation (see, e.g., U.S. Pat.Nos. 4,044,126, 4,414,209 and 4,364,923, which describe aerosols fordelivery of a steroid useful for treatment of inflammatory diseases,particularly asthma and are herein incorporated by reference in theirentireties). These formulations for administration to the respiratorytract can be in the form of an aerosol or solution for a nebulizer, oras a microfine powder for insufflations, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation will, in one embodiment, have diameters of less than 50microns, in one embodiment less than 10 microns.

An anti-CTLA-4 antibody described herein can be formulated for local ortopical application, such as for topical application to the skin andmucous membranes, such as in the eye, in the form of gels, creams, andlotions and for application to the eye or for intracisternal orintraspinal application. Topical administration is contemplated fortransdermal delivery and also for administration to the eyes or mucosa,or for inhalation therapies. Nasal solutions of the antibody alone or incombination with other pharmaceutically acceptable excipients can alsobe administered.

Transdermal patches, including iontophoretic and electrophoreticdevices, are well known to those of skill in the art, and can be used toadminister an antibody. For example, such patches are disclosed in U.S.Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975,6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957, all of whichare herein incorporated by reference in their entireties.

In certain embodiments, a pharmaceutical composition comprising anantibody or antigen-binding fragment thereof described herein is alyophilized powder, which can be reconstituted for administration assolutions, emulsions and other mixtures. It may also be reconstitutedand formulated as solids or gels. The lyophilized powder is prepared bydissolving an antibody or antigen-binding fragment thereof describedherein, or a pharmaceutically acceptable derivative thereof, in asuitable solvent. In some embodiments, the lyophilized powder issterile. The solvent may contain an excipient which improves thestability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbitol, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

The anti-CTLA-4 antibodies described herein and other compositionsprovided herein can also be formulated to be targeted to a particulartissue, receptor, or other area of the body of the subject to betreated. Many such targeting methods are well known to those of skill inthe art. All such targeting methods are contemplated herein for use inthe instant compositions. For non-limiting examples of targetingmethods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359,6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082,6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252,5,840,674, 5,759,542 and 5,709,874, all of which are herein incorporatedby reference in their entireties. In a specific embodiment, an antibodyor antigen-binding fragment thereof described herein is targeted to atumor.

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

6.4 Methods of Use and Uses

In another aspect, the instant disclosure provides a method of treatinga subject using the anti-CTLA-4 antibodies described herein. Any diseaseor disorder in a subject that would benefit from inhibition of CTLA-4function can be treated using the anti-CTLA-4 antibodies describedherein. The anti-CTLA-4 antibodies described herein are particularlyuseful for inhibiting immune system tolerance to tumors, and accordinglycan be used as an immunotherapy for subjects with cancer. For example,in certain embodiments, the instant disclosure provides a method ofincreasing T-cell activation in response to an antigen in a subject, themethod comprising administering to the subject an effective amount of ananti-CTLA-4 antibody or pharmaceutical composition thereof, as describedherein. In certain embodiments, the instant disclosure provides a methodof treating cancer in a subject, the method comprising administering tothe subject an effective amount of the antibody or pharmaceuticalcomposition, as described herein.

Cancers that can be treated with the antibodies, therapeuticcombinations, or pharmaceutical compositions described herein include,without limitation, solid cancer (e.g., relapsed or refractory solidcancer, and advanced or metastatic solid cancer), carcinoma, sarcoma,melanoma (e.g., stage III or stage IV melanoma), small cell lung cancer,non-small cell lung cancer, urothelial cancer, ovarian cancer, prostatecancer (e.g., metastatic hormone-refractory prostate cancer andprogressive metastatic prostate cancer), pancreatic cancer, breastcancer (e.g., HER2′ breast cancer (e.g., relapsed/refractory HER2′breast cancer)), head and neck cancer (e.g., relapsed/refractory headand neck squamous cell carcinoma (HNSCC)), glioma, malignant glioma,glioblastoma multiforme, brain metastasis, merkel cancer, gastriccancer, gastroesophageal cancer, renal cell carcinoma, uveal melanoma,colon cancer, cervical cancer, lymphoma (e.g., relapsed or refractorylymphoma), non-Hodgkin's lymphoma, Hodgkin's lymphoma, leukemia, andmultiple myeloma. In certain embodiments, the cancer is treated withintratumoral administration of an antibody, therapeutic combination, orpharmaceutical composition described herein. Cancers that can be treatedwith intratumoral administration of the antibodies, therapeuticcombinations, or pharmaceutical compositions described herein include,without limitation, solid tumors (e.g., advanced or metastatic solidtumors), head and neck cancer (e.g., relapsed/refractory head and necksquamous cell carcinoma (HNSCC)), and breast cancer (e.g., HER2′ breastcancer (e.g., relapsed/refractory HER2′ breast cancer)).

In certain embodiments, the cancer treated in accordance with themethods described herein is a solid tumor. In certain embodiments, thecancer treated in accordance with the methods described herein is ametastatic or locally advanced cancer (e.g., a metastatic or locallyadvanced solid tumor). In certain embodiments, the cancer is treated inaccordance with a method described herein as a first cancer therapyafter diagnosis of the metastatic or locally advanced tumor (e.g.,within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1,2, 3, 4, 6, 8, or 12 months after diagnosis). In certain embodiments,the cancer is treated in accordance with a method described herein asthe first cancer therapy after diagnosis of tumor progression (e.g.,within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1,2, 3, 4, 6, 8, or 12 months after diagnosis of tumor progression) thathas occurred despite previous treatment of the tumor with a differentcancer therapy, optionally wherein the method described herein isprovided as the second cancer therapy administered. In certainembodiments, the cancer is treated in accordance with a method describedherein as the first cancer therapy after diagnosis of toxicity of adifferent cancer therapy (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2,3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months afterdiagnosis of toxicity of the different cancer therapy), optionallywherein the method described herein is provided as the second cancertherapy administered. In certain embodiments, the cancer treated inaccordance with the methods described herein is a metastatic or locallyadvanced cancer (e.g., solid tumor) for which no standard therapy isavailable. In other embodiments, the cancer treated in accordance withthe methods described herein is a metastatic or locally advanced cancer(e.g., solid tumor) for which a standard therapy has failed (i.e., thecancer has progressed after the standard therapy). In certainembodiments, a therapy fails if the cancer is refractory to the therapy.In certain embodiments, a therapy fails if the cancer relapses afterresponding, fully or partially, to the therapy. In certain embodiments,metastatic or locally advanced cancer (e.g., solid tumor) has beenconfirmed histologically or cytologically.

In certain embodiments, the cancer is a solid tumor. In certainembodiments, the cancer (e.g., solid tumor) expresses PD-L1. In certainembodiments, the percentage of tumor cells in a sample of the cancer(e.g., solid tumor) that exhibit detectable expression (e.g., partial orcomplete expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%).In certain embodiments, the percentage of tumor cells in a sample of thecancer (e.g., solid tumor) that exhibit detectable membrane expression(e.g., partial or complete membrane expression) of PD-L1 is at least 1%(e.g., at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 60%, 70%, 80%, or 90%). In certain embodiments, the percentage oftumor cells in a sample of the cancer (e.g., solid tumor) that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 1%. In certain embodiments, thepercentage of tumor cells in a sample of the cancer (e.g., solid tumor)that exhibit detectable membrane expression (e.g., partial or completemembrane expression) of PD-L1 is at least 5%. In certain embodiments,the percentage of tumor cells in a sample of the cancer (e.g., solidtumor) that exhibit detectable membrane expression (e.g., partial orcomplete membrane expression) of PD-L1 is at least 25%. In certainembodiments, the percentage of tumor cells in a sample of the cancer(e.g., solid tumor) that exhibit detectable membrane expression (e.g.,partial or complete membrane expression) of PD-L1 is at least 50%.

In certain embodiments, the metastatic or locally advanced cancer (e.g.,solid tumor) expresses PD-L1. In certain embodiments, the percentage oftumor cells in a sample of the metastatic or locally advanced cancer(e.g., solid tumor) that exhibit detectable expression (e.g., partial orcomplete expression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%).In certain embodiments, the percentage of tumor cells in a sample of themetastatic or locally advanced cancer (e.g., solid tumor) that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 1% (e.g., at least 2%, 3%, 4%, 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%). Incertain embodiments, the percentage of tumor cells in a sample of themetastatic or locally advanced cancer (e.g., solid tumor) that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 1%. In certain embodiments, thepercentage of tumor cells in a sample of the metastatic or locallyadvanced cancer (e.g., solid tumor) that exhibit detectable membraneexpression (e.g., partial or complete membrane expression) of PD-L1 isat least 5%. In certain embodiments, the percentage of tumor cells in asample of the metastatic or locally advanced cancer (e.g., solid tumor)that exhibit detectable membrane expression (e.g., partial or completemembrane expression) of PD-L1 is at least 25%. In certain embodiments,the percentage of tumor cells in a sample of the metastatic or locallyadvanced cancer (e.g., solid tumor) that exhibit detectable membraneexpression (e.g., partial or complete membrane expression) of PD-L1 isat least 50%.

For each and every one of the methods described herein that requires acertain percentage of cells in a sample exhibit detectable expression(e.g., membrane expression, partial or complete membrane expression) ofPD-L1, the expression of PD-L1 can be detected by any method well knownin the art, including but not limited to immunohistochemistry. Exemplaryimmunohistochemistry assays for measuring PD-L1 expression in tumorcells are provided in Hirsch et al. (2017, J. Thoracic Oncol. 12(2):208-222), Rimm et al. (2017, JAMA Oncol. 3(8): 1051-1058), and Diggs andHsueh (2017, Biomarker Res. 5:12), which are incorporated by referenceherein in their entirety.

In certain embodiments, the cancer treated in accordance with a methoddescribed herein is a metastatic or locally advanced non-small cell lungcancer (NSCLC). In certain embodiments, the cancer treated in accordancewith a method described herein is a metastatic non-small cell lungcancer (NSCLC). In certain embodiments, the cancer treated in accordancewith a method described herein is a Stage IV, metastatic or locallyadvanced NSCLC. In certain embodiments, the cancer treated in accordancewith a method described herein is a Stage IV, metastatic NSCLC. Incertain embodiments, the percentage of tumor cells in a sample of themetastatic or locally advanced NSCLC that exhibit detectable expression(e.g., partial or complete expression) of PD-L1 is at least 1%, 2%, 3%,4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or90%. In certain embodiments, the percentage of tumor cells in a sampleof the metastatic or locally advanced NSCLC that exhibit detectablemembrane expression (e.g., partial or complete membrane expression) ofPD-L1 is at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 60%, 70%, 80%, or 90%. In certain embodiments, the percentageof tumor cells in a sample of the metastatic or locally advanced NSCLCthat exhibit detectable membrane expression (e.g., partial or completemembrane expression) of PD-L1 is at least 1%. In certain embodiments,the percentage of tumor cells in a sample of the metastatic or locallyadvanced NSCLC that exhibit detectable membrane expression (e.g.,partial or complete membrane expression) of PD-L1 is at least 5%. Incertain embodiments, the percentage of tumor cells in a sample of themetastatic or locally advanced NSCLC that exhibit detectable membraneexpression (e.g., partial or complete membrane expression) of PD-L1 isat least 25%. In certain embodiments, the percentage of tumor cells in asample of the metastatic or locally advanced NSCLC that exhibitdetectable membrane expression (e.g., partial or complete membraneexpression) of PD-L1 is at least 50%. In certain embodiments, themetastatic or locally advanced NSCLC has no EGFR or ALK genomic tumoraberrations. In certain embodiments, the metastatic or locally advancedNSCLC has no EGFR sensitizing mutation (e.g., mutation that is amenableto treatment with a tyrosine kinase inhibitor including erlotinib,gefitinib, or afatanib) or ALK translocation. In certain embodiments,the subject having the metastatic or locally advanced NSCLC has receivedno prior systemic chemotherapy treatment for metastatic or locallyadvanced NSCLC. In certain embodiments, the metastatic or locallyadvanced NSCLC is treated in accordance with a method described hereinas a first cancer therapy after diagnosis (e.g., within 1, 2, 3, 4, 5,or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12months after diagnosis) of the metastatic or locally advanced NSCLC. Incertain embodiments, the method comprises treating a subject havingNSCLC (e.g., Stage IV, metastatic, or locally advanced NSCLC) using ananti-CTLA-4 antibody described herein, e.g., AGEN1884.H3 (IgG₁S239D/A330L/I332E), or pharmaceutical composition comprising suchanti-CTLA-4 antibody, wherein the percentage of tumor cells in a sampleof the NSCLC that exhibit detectable membrane expression (e.g., partialor complete membrane expression) of PD-L1 is at least 1%, 2%, 3%, 4%,5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90%,and wherein the method is provided as a first cancer therapy afterdiagnosis of the cervical cancer (e.g., within 1, 2, 3, 4, 5, or 6 days;1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months afterdiagnosis).

In certain embodiments, the cancer treated in accordance with themethods described herein is a cervical cancer. In certain embodiments,the cancer treated in accordance with the methods described herein is ametastatic or locally advanced, unresectable squamous cell carcinoma,adenosquamous carcinoma, or adenocarcinoma of the cervix. In certainembodiments, the cancer treated in accordance with the methods describedherein is an unresectable or metastatic cervical cancer. In certainembodiments, the cervical cancer has progressed after a standard therapy(e.g., has relapsed after the standard therapy, or is refractory to thestandard therapy). In certain embodiments, the standard therapycomprises a platinum-containing chemotherapy. In certain embodiments,the platinum-containing chemotherapy is selected from the groupconsisting of cisplatin, carboplatin, oxaliplatin, nedaplatin,satraplatin, picoplatin, triplatin, phenanthriplatin, iproplatin,lobapatin, heptaplatin, lipoplatin, and a combination thereof. Incertain embodiments, the standard therapy further comprises a secondchemotherapy. In certain embodiments, the second chemotherapy isselected from the group consisting of a nucleotide analog (e.g.,gemcitabine), a folate antimetabolite (e.g., pemetrexed), and a taxane(e.g., paclitaxel). In certain embodiments, the standard therapy is anyplatinum-based doublet chemotherapy (PT-DC) (also known asplatinum-containing doublet) known in the art. In certain embodiments,the PT-DC comprises cisplatin and gemcitabine, cisplatin and pemetrexed,cisplatin and paclitaxel, carboplatin and paclitaxel, or cisplatin andtopotecan. The standard therapy (e.g., one comprising a PT-DC) canoptionally further comprise one or more additional therapies, such asbevacizumab. In certain embodiments, the standard therapy comprisespaclitaxel and topotecan. In certain embodiments, the cervical cancer isHPV positive. In certain embodiments, the cervical cancer is associatedwith microsatellite instability. In certain embodiments, the cancertreated in accordance with the methods described herein is a metastaticor locally advanced, unresectable squamous cell carcinoma, adenosquamouscarcinoma, or adenocarcinoma of the cervix that has relapsed after aplatinum-containing doublet administered for treatment of advanced(recurrent, unresectable, or metastatic) disease. In certainembodiments, the cancer of the cervix is treated in accordance with amethod described herein as a first cancer therapy after diagnosis of thecervical cancer (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6,8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis). Incertain embodiments, the cancer of the cervix is treated in accordancewith a method described herein as the first cancer therapy afterdiagnosis of tumor progression (e.g., within 1, 2, 3, 4, 5, or 6 days;1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months afterdiagnosis of tumor progression) that has occurred despite previoustreatment of the cancer of the cervix with a different cancer therapy,optionally wherein the method described herein is provided as the secondcancer therapy administered. In certain embodiments, the cancer of thecervix is treated in accordance with a method described herein as thefirst cancer therapy after diagnosis of toxicity of a different cancertherapy (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of toxicity ofthe different cancer therapy), optionally wherein the method describedherein is provided as the second cancer therapy administered. In certainembodiments, the method comprises treating a subject having cervicalcancer (e.g., a metastatic or locally advanced, unresectable squamouscell carcinoma, adenosquamous carcinoma, or adenocarcinoma of thecervix) using an anti-CTLA-4 antibody described herein, e.g.,AGEN1884.H3 (IgG₁ S239D/A330L/I332E), or pharmaceutical compositioncomprising such anti-CTLA-4 antibody, wherein the method is provided asa first cancer therapy after diagnosis of the cervical cancer (e.g.,within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1,2, 3, 4, 6, 8, or 12 months after diagnosis). In certain embodiments,the method comprises treating a subject having cervical cancer (e.g., ametastatic or locally advanced, unresectable squamous cell carcinoma,adenosquamous carcinoma, or adenocarcinoma of the cervix) using ananti-CTLA-4 antibody described herein, e.g., AGEN1884.H3 (IgG₁S239D/A330L/I332E), or pharmaceutical composition comprising suchanti-CTLA-4 antibody, wherein the method is provided after diagnosis oftumor progression (e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6,8, or 12 weeks; or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis oftumor progression) that has occurred despite previous treatment of thecervical cancer with a different cancer therapy, or provided afterdiagnosis of toxicity of a different cancer therapy (e.g., within 1, 2,3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4, 6, 8,or 12 months after diagnosis of toxicity of the different cancertherapy), and wherein the method described herein is provided as thesecond cancer therapy administered.

In certain embodiments, the cancer treated in accordance with themethods described herein is a cutaneous squamous-cell carcinoma (cSCC).In certain embodiments, the cancer treated in accordance with themethods described herein is a Stage IV cutaneous squamous-cell carcinoma(cSCC). In certain embodiments, the cSCC (e.g., Stage IV cSCC) is notcurable with radiation therapy. In certain embodiments, the Stage IVcSCC is diagnosed histologically or cytologically according to theeighth edition of the American Joint Committee on Cancer staging manual(AJCC-8). In certain embodiments, the cSCC (e.g., Stage IV cSCC) istreated in accordance with a method described herein as a first cancertherapy after diagnosis of the cSCC (e.g., Stage IV cSCC) (e.g., within1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4,6, 8, or 12 months after diagnosis). In certain embodiments, the cSCC(e.g., Stage IV cSCC) is treated in accordance with a method describedherein as the first cancer therapy after diagnosis of tumor progression(e.g., within 1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks;or, 1, 2, 3, 4, 6, 8, or 12 months after diagnosis of tumor progression)that has occurred despite previous treatment of the cSCC (e.g., Stage IVcSCC) with a different cancer therapy, optionally wherein the methoddescribed herein is provided as the second cancer therapy administered.In certain embodiments, the cSCC (e.g., Stage IV cSCC) is treated inaccording with a method described herein as the first cancer therapyafter diagnosis of toxicity of a different cancer therapy (e.g., within1, 2, 3, 4, 5, or 6 days; 1, 2, 3, 4, 6, 8, or 12 weeks; or, 1, 2, 3, 4,6, 8, or 12 months after diagnosis of toxicity of the different cancertherapy), optionally wherein the method described herein is provided asthe second cancer therapy administered.

In certain embodiments, the cancer treated in accordance with themethods described herein is B cell lymphoma (e.g., B cell chroniclymphocytic leukemia, B cell non-Hodgkin lymphoma, cutaneous B celllymphoma, diffuse large B cell lymphoma), basal cell carcinoma, bladdercancer, blastoma, brain metastasis, breast cancer, Burkitt lymphoma,carcinoma (e.g., adenocarcinoma (e.g., of the gastroesophagealjunction)), cervical cancer, colon cancer, colorectal cancer (coloncancer and rectal cancer), endometrial carcinoma, esophageal cancer,Ewing sarcoma, follicular lymphoma, gastric cancer, gastroesophagealjunction carcinoma, gastrointestinal cancer, glioblastoma (e.g.,glioblastoma multiforme, e.g., newly diagnosed or recurrent), glioma,head and neck cancer (e.g., head and neck squamous cell carcinoma),hepatic metastasis, Hodgkin's and non-Hodgkin's lymphoma, kidney cancer(e.g., renal cell carcinoma and Wilms' tumors), laryngeal cancer,leukemia (e.g., chronic myelocytic leukemia, hairy cell leukemia), livercancer (e.g., hepatic carcinoma and hepatoma), lung cancer (e.g.,non-small cell lung cancer and small-cell lung cancer), lymphblasticlymphoma, lymphoma, mantle cell lymphoma, metastatic brain tumor,metastatic cancer, myeloma (e.g., multiple myeloma), neuroblastoma,ocular melanoma, oropharyngeal cancer, osteosarcoma, ovarian cancer,pancreatic cancer (e.g., pancreatis ductal adenocarcinoma), prostatecancer (e.g., hormone refractory (e.g., castration resistant),metastatic, metastatic hormone refractory (e.g., castration resistant,androgen independent)), renal cell carcinoma (e.g., metastatic),salivary gland carcinoma, sarcoma (e.g., rhabdomyosarcoma), skin cancer(e.g., melanoma (e.g., metastatic melanoma)), soft tissue sarcoma, solidtumor, squamous cell carcinoma, synovia sarcoma, testicular cancer,thyroid cancer, transitional cell cancer (urothelial cell cancer), uvealmelanoma (e.g., metastatic), verrucous carcinoma, vulval cancer, andWaldenstrom macroglobulinemia.

In certain embodiments, the cancer treated in accordance with themethods described herein is human sarcoma or carcinoma, e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endothelio sarcoma, lymphangiosarcoma,lymphangioendothelio sarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma (e.g., metastatic), hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, testicular tumor, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, glioblastomamultiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma,pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma,meningioma, melanoma, neuroblastoma, or retinoblastoma.

In certain embodiments, the cancer treated in accordance with themethods described herein is angio sarcoma.

In certain embodiments, the cancer treated in accordance with themethods described herein is an acute lymphocytic leukemia or acutemyelocytic leukemia (e.g., myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia or chronic lymphocytic leukemia); Hodgkin'sdisease; non-Hodgkin's disease; acute myeloid leukemia; B-cell lymphoma;T-cell lymphoma; anaplastic large cell lymphoma; intraocular lymphoma;follicular lymphoma; small intestine lymphoma; or orsplenic marginalzone lymphoma.

In certain embodiments, the cancer treated in accordance with themethods described herein is multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, gastrointestinal stromal tumors,head and/or neck cancer (e.g., squamous cell carcinoma of thehypopharynx, squamous cell carcinoma of the larynx, cell carcinoma ofthe oropharynx, or verrucous carcinoma of the larynx), endometrialstromal sarcoma, mast cell sarcoma, adult soft tissue sarcoma, uterinesarcoma, merkel cell carcinoma, urothelial carcinoma, melanoma withbrain metastases, uveal melanoma, uveal melanoma with liver metastases,non-small cell lung cancer, rectal cancer, or myelodysplastic syndrome.In some embodiments, the cancer treated in accordance with the methodsis metastatic.

In certain embodiments, the cancer treated in accordance with themethods described herein is prostate cancer, breast cancer, lung cancer,colorectal cancer, melanoma, bronchial cancer, bladder cancer, brain orcentral nervous system cancer, peripheral nervous system cancer, uterineor endometrial cancer, cancer of the oral cavity or pharynx,non-Hodgkin's lymphoma, thyroid cancer, kidney cancer, biliary tractcancer, small bowel or appendix cancer, salivary gland cancer, thyroidgland cancer, adrenal gland cancer, squamous cell cancer, mesothelioma,osteocarcinoma, thyoma/thymic carcinoma, glioblastoma, myelodysplasticsyndrome, soft tissue sarcoma, DIPG, adenocarcinoma, osteosarcoma,chondrosarcoma, leukemia, or pancreatic cancer. In some embodiments, thecancer treated in accordance with the methods described herein includesa carcinoma (e.g., an adenocarcinoma), lymphoma, blastoma, melanoma,sarcoma, or leukemia.

In certain embodiments, the cancer treated in accordance with themethods described herein is squamous cell cancer, small-cell lungcancer, non-small cell lung cancer, gastrointestinal cancer, Hodgkin'slymphoma, non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma,glioma, cervical cancer, ovarian cancer, liver cancer (e.g., hepaticcarcinoma and hepatoma), bladder cancer, breast cancer, inflammatorybreast cancer, Merkel cell carcinoma, colon cancer, colorectal cancer,stomach cancer, urinary bladder cancer, endometrial carcinoma, myeloma(e.g., multiple myeloma), salivary gland, carcinoma, kidney cancer(e.g., renal cell carcinoma and Wilms' tumors), basal cell carcinoma,melanoma, prostate cancer, vulval cancer, thyroid cancer, testicularcancer, esophageal cancer, serous adenocarcinoma or various types ofhead and neck cancer. In certain embodiments, the cancer treated inaccordance with the methods described herein includes desmoplasticmelanoma, inflammatory breast cancer, thymoma, rectal cancer, analcancer, or surgically treatable or non-surgically treatable brain stemglioma. In a specific embodiment, the cancer is a solid tumor. Inanother specific embodiment, the cancer is glioblastoma multiforme. Insome embodiments, the glioblastoma multiforme is recurrent. In someembodiments, the glioblastoma multiforme is newly diagnosed. In someembodiments, the glioblastoma multiforme is in a subject havingnon-methylated MGMT promoters. In some embodiments, the glioblastomamultiforme is refractory to Bevacizumab therapy. In some embodiments,the glioblastoma multiforme is in a subject that has not receivedBevacizumab therapy.

In certain embodiments, the cancer treated in accordance with themethods described herein is metastatic melanoma (e.g., resistantmetastatic melanoma), metastatic ovarian cancer, or metastatic renalcell carcinoma. In certain embodiments, the cancer treated in accordancewith the methods described herein is melanoma that is resistant toIpilimumab. In some embodiments, the cancer treated in accordance withthe methods described herein is melanoma that is resistant to Nivolumabor Pembrolizumab. In some embodiments, the cancer treated in accordancewith the methods described herein is melanoma that is resistant toIpilimumab and Nivolumab or Pembrolizumab.

In certain embodiments, the cancer treated in accordance with themethods described herein is breast cancer (e.g., herceptin resistantbreast cancer and trastuzumab-DM1 (T-DM1) resistant breast cancer),prostate cancer, glioblastoma multiforme, colorectal cancer, sarcoma,bladder cancer, cervical cancer, HPV-associated cancers, cancers of thevagina, cancers of the vulva, cancers of the penis, cancer of the anus,cancer of the rectum, cancer of the oropharynx, multiple myeloma, renalcell carcinoma, ovarian cancer, hepatocellular cancer, endometrialcancer, pancreatic cancer, lymphoma, and leukemia (e.g., elderlyleukemia, acute myeloid leukemia (AML), and elderly AML).

In certain embodiments, the cancer treated in accordance with themethods described herein is metastatic malignant melanoma (e.g.,cutaneous or intraocular malignant melanoma), renal cancer (e.g., clearcell carcinoma), prostate cancer (e.g., hormone refractory prostateadenocarcinoma), breast cancer, colon cancer, lung cancer (e.g.,non-small cell lung cancer), bone cancer, pancreatic cancer, skincancer, cancer of the head or neck, uterine cancer, ovarian cancer,rectal cancer, cancer of the anal region, stomach cancer, testicularcancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma ofthe endometrium, carcinoma of the cervix, carcinoma of the vagina,carcinoma of the vulva, cancer of the esophagus, cancer of the smallintestine, cancer of the endocrine system, cancer of the thyroid gland,cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma ofsoft tissue, cancer of the urethra, cancer of the penis, chronic oracute leukemias including acute myeloid leukemia, chronic myeloidleukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia,solid tumors of childhood, lymphocytic lymphoma, cancer of the bladder,cancer of the kidney or ureter, carcinoma of the renal pelvis, neoplasmof the central nervous system (CNS), primary CNS lymphoma, tumorangiogenesis, spinal axis tumor, brain stem glioma, glioma, pituitaryadenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell cancer,T-cell lymphoma, environmentally induced cancers including those inducedby asbestos, esophageal cancer, liver cancer, refractory or recurrentmalignancies, metastatic cancers, cancers that express PD-L1, andcombinations of said cancers.

In certain embodiments, the subject has previously received animmunotherapy. In certain embodiments, the subject has not previouslyreceived any immunotherapy. In certain embodiments, the cancer is anadvanced or metastatic cancer.

In certain embodiments, the instant disclosure provides a method ofpreventing or treating an infectious disease in a subject, the methodcomprising administering to the subject an effective amount of ananti-CTLA-4 antibody or pharmaceutical composition thereof, as describedherein. In one embodiment, provided herein are methods for preventingand/or treating an infection (e.g., a viral infection, a bacterialinfection, a fungal infection, a protozoal infection, or a parasiticinfection). The infection prevented and/or treated in accordance withthe methods can be caused by an infectious agent identified herein. In aspecific embodiment, an anti-CTLA-4 antibody described herein or acomposition thereof is the only active agent administered to a subject.In some embodiments, an anti-CTLA-4 antibody described herein or acomposition thereof is used in combination with anti-infectiveinterventions (e.g., antivirals, antibacterials, antifungals, oranti-helminthics) for the treatment of infectious diseases.

Infectious diseases that can be treated and/or prevented by anti-CTLA-4antibodies or pharmaceutical compositions described herein are caused byinfectious agents including but not limited to bacteria, parasites,fungi, protozae, and viruses. In a specific embodiment, the infectiousdisease treated and/or prevented by anti-CTLA-4 antibodies orpharmaceutical compositions described herein is caused by a virus. Viraldiseases or viral infections that can be prevented and/or treated inaccordance with the methods described herein include, but are notlimited to, those caused by hepatitis type A, hepatitis type B,hepatitis type C, influenza (e.g., influenza A or influenza B),varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, small pox,Epstein Barr virus, human immunodeficiency virus type I (HIV-I), humanimmunodeficiency virus type II (HIV-II), and agents of viral diseasessuch as viral meningitis, encephalitis, dengue or small pox.

Bacterial infections that can be prevented and/or treated includeinfections caused by Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa. Bacterial diseasescaused by bacteria (e.g., Escherichia coli, Klebsiella pneumoniae,Staphylococcus aureus, Enterococcus faecalis, Proteus vulgaris,Staphylococcus viridans, and Pseudomonas aeruginosa) that can beprevented and/or treated in accordance with the methods described hereininclude, but are not limited to, Mycobacteria rickettsia, Mycoplasma,Neisseria, S. pneumonia, Borrelia burgdorferi (Lyme disease), Bacillusantracis (anthrax), tetanus, Streptococcus, Staphylococcus,mycobacterium, pertissus, cholera, plague, diptheria, chlamydia, S.aureus and legionella.

Protozoal diseases or protozoal infections caused by protozoa that canbe prevented and/or treated in accordance with the methods describedherein include, but are not limited to, leishmania, coccidiosis,trypanosoma schistosoma or malaria. Parasitic diseases or parasiticinfections caused by parasites that can be prevented and/or treated inaccordance with the methods described herein include, but are notlimited to, chlamydia and rickettsia.

Fungal diseases or fungal infections that can be prevented and/ortreated in accordance with the methods described herein include, but arenot limited to, those caused by Candida infections, zygomycosis, Candidamastitis, progressive disseminated trichosporonosis with latenttrichosporonemia, disseminated candidiasis, pulmonaryparacoccidioidomycosis, pulmonary aspergillosis, Pneumocystis cariniipneumonia, cryptococcal meningitis, coccidioidal meningoencephalitis andcerebrospinal vasculitis, Aspergillus niger infection, Fusariumkeratitis, paranasal sinus mycoses, Aspergillus fumigatus endocarditis,tibial dyschondroplasia, Candida glabrata vaginitis, oropharyngealcandidiasis, X-linked chronic granulomatous disease, tinea pedis,cutaneous candidiasis, mycotic placentitis, disseminatedtrichosporonosis, allergic bronchopulmonary aspergillosis, mycotickeratitis, Cryptococcus neoformans infection, fungal peritonitis,Curvularia geniculata infection, staphylococcal endophthalmitis,sporotrichosis, and dermatophytosis.

In certain embodiments, the infectious disease is acute. In certainembodiments, the infectious disease is chronic. In certain embodiments,the infectious disease is caused by flavivirus, e.g., West Nile virus,Saint Louis encephalitis virus, Powassan virus, tick-borne encephalitisvirus, dengue virus, zika virus, Kyasanur Forest disease virus, yellowfever virus, and chikungunya virus. In certain embodiments, theinfectious disease is caused by Ebola virus. In certain embodiments, theinfectious disease is caused by influenza virus. In certain embodiments,the infectious disease is caused by Human Immunodeficiency Virus (HIV),Hepatitis B virus (HBV) or Hepatitis C virus (HCV). In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositionthereof, as described herein, promotes viral control. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositionthereof, as described herein, eliminates viral reservoirs.

The present invention relates in one aspect to an anti-CTLA-4 antibodyof the invention and/or a pharmaceutical composition of the inventioncomprising an anti-CTLA-4 antibody of the invention and apharmaceutically acceptable carrier or excipient, for use as amedicament.

The present invention relates, in one aspect, to an anti-CTLA-4 antibodyof the invention, and/or its use in combination with pharmaceuticallyacceptable carriers or excipients, for preparing pharmaceuticalcompositions or medicaments for immunotherapy (e.g., an immunotherapyfor increasing T-cell activation in response to an antigen in a subject,treating cancer, or treating or preventing infectious diseases).

The present invention relates in one aspect to an anti-CTLA-4 antibodyof the invention and/or a pharmaceutical composition of the inventioncomprising an anti-CTLA-4 antibody of the invention and apharmaceutically acceptable carrier or excipient, for use in a methodfor the treatment of cancer.

The present invention relates in one aspect to an anti-CTLA-4 antibodyof the invention and/or a pharmaceutical composition of the inventioncomprising an anti-CTLA-4 antibody of the invention and apharmaceutically acceptable carrier or excipient, for use in a methodfor inhibiting immune system tolerance to tumors and/or forimmunotherapy for subjects with cancer.

The present invention relates in one aspect to an anti-CTLA-4 antibodyof the invention and/or a pharmaceutical composition of the inventioncomprising an anti-CTLA-4 antibody of the invention and apharmaceutically acceptable carrier or excipient, for use in a methodfor the treatment of an infectious disease.

In certain embodiments, the anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered as a monotherapy.

In certain embodiments, these methods further comprise administering anadditional therapeutic agent to the subject. In certain embodiments, theadditional therapeutic agent is a chemotherapeutic or a checkpointtargeting agent. In certain embodiments, the checkpoint targeting agentis selected from the group consisting of an antagonist anti-PD-1antibody, an antagonist anti-PD-L1 antibody, an antagonist anti-PD-L2antibody, an antagonist anti-CTLA-4 antibody, an antagonist anti-TIM-3antibody, an antagonist anti-LAG-3 antibody, an antagonist anti-CEACAM1antibody, an agonist anti-GITR antibody, an agonist anti-OX40 antibody,an agonist anti-CD137 antibody, an agonist anti-DR3 antibody, an agonistanti-TNFSF14 antibody, and an agonist anti-CD27 antibody. In certainembodiments, the checkpoint targeting agent is an antagonist anti-PD-1antibody. In certain embodiments, the checkpoint targeting agent is anantagonist anti-PD-L1 antibody. In certain embodiments, the checkpointtargeting agent is an antagonist anti-LAG-3 antibody. In certainembodiments, the additional therapeutic agent is an agonist to a tumornecrosis factor receptor superfamily member or a tumor necrosis factorsuperfamily member.

In certain embodiments, the present invention relates to (a) ananti-CTLA-4 antibody of the invention and/or a pharmaceuticalcomposition of the invention comprising an anti-CTLA-4 antibody of theinvention and a pharmaceutically acceptable carrier or excipient and (b)an additional therapeutic agent, for use as a medicament. In a preferredembodiment, the additional therapeutic agent is a chemotherapeutic or acheckpoint targeting agent.

In certain embodiments, the present invention relates to (a) ananti-CTLA-4 antibody of the invention and/or a pharmaceuticalcomposition of the invention comprising an anti-CTLA-4 antibody of theinvention and a pharmaceutically acceptable carrier or excipient and (b)an additional therapeutic agent, for use in a method for the treatmentof cancer.

In certain embodiments, the present invention relates to (a) ananti-CTLA-4 antibody of the invention and/or a pharmaceuticalcomposition of the invention comprising an anti-CTLA-4 antibody of theinvention and a pharmaceutically acceptable carrier or excipient and (b)an additional therapeutic agent, for use in a method for the treatmentof an infectious disease.

In certain embodiments, an anti-CTLA-4 antibody described herein isadministered to a subject in combination with a compound that targets animmunomodulatory enzyme(s) such as IDO (indoleamine-(2,3)-dioxygenase)and/or TDO (tryptophan 2,3-dioxygenase). In certain embodiments, suchcompound is selected from the group consisting of epacadostat (IncyteCorp; see, e.g., WO 2010/005958 which is incorporated by referenceherein in its entirety), F001287 (Flexus Biosciences), indoximod(NewLink Genetics), and NLG919 (NewLink Genetics). In one embodiment,the compound is epacadostat. In another embodiment, the compound isF001287. In another embodiment, the compound is indoximod. In anotherembodiment, the compound is NLG919.

In certain embodiments, the present invention relates to (a) ananti-CTLA-4 antibody of the invention and/or a pharmaceuticalcomposition of the invention comprising an anti-CTLA-4 antibody of theinvention and a pharmaceutically acceptable carrier or excipient and (b)a compound that targets an immunomodulatory enzyme, for use as amedicament. In a preferred embodiment, the compound targets IDO and/orTDO.

In certain embodiments, the present invention relates to (a) ananti-CTLA-4 antibody of the invention and/or a pharmaceuticalcomposition of the invention comprising an anti-CTLA-4 antibody of theinvention and a pharmaceutically acceptable carrier or excipient and (b)a compound that targets an immunomodulatory enzyme, for use in a methodfor the treatment of cancer. In a preferred embodiment, the compoundtargets IDO and/or TDO.

In certain embodiments, an anti-CTLA-4 antibody described herein isadministered to a subject in combination with a vaccine. In certainembodiments, the vaccine is a heat shock protein based tumor vaccine ora heat shock protein based pathogen vaccine. In a specific embodiment,an anti-CTLA-4 antibody described herein is administered to a subject incombination with a heat shock protein based tumor-vaccine. Heat shockproteins (HSPs) are a family of highly conserved proteins foundubiquitously across all species. Their expression can be powerfullyinduced to much higher levels as a result of heat shock or other formsof stress, including exposure to toxins, oxidative stress or glucosedeprivation. Five families have been classified according to molecularweight: HSP-110, -90, -70, -60 and -28. HSPs deliver immunogenicpeptides through the cross-presentation pathway in antigen presentingcells (APCs) such as macrophages and dendritic cells (DCs), leading to Tcell activation. HSPs function as chaperone carriers of tumor-associatedantigenic peptides forming complexes able to induce tumor-specificimmunity. Upon release from dying tumor cells, the HSP-antigen complexesare taken up by antigen-presenting cells (APCs) wherein the antigens areprocessed into peptides that bind MHC class I and class II moleculesleading to the activation of anti-tumor CD8+ and CD4+ T cells. Theimmunity elicited by HSP complexes derived from tumor preparations isspecifically directed against the unique antigenic peptide repertoireexpressed by the cancer of each subject.

A heat shock protein peptide complex (HSPPC) is a protein peptidecomplex consisting of a heat shock protein non-covalently complexed withantigenic peptides. HSPPCs elicit both innate and adaptive immuneresponses. In a specific embodiment, the antigenic peptide(s) displaysantigenicity for the cancer being treated. HSPPCs are efficiently seizedby APCs via membrane receptors (mainly CD91) or by binding to Toll-likereceptors. HSPPC internalization results in functional maturation of theAPCs with chemokine and cytokine production leading to activation ofnatural killer cells (NK), monocytes and Th1 and Th-2-mediated immuneresponses. In certain embodiments, HSPPCs used in methods describedherein comprise one or more heat shock proteins from the hsp60, hsp70,or hsp90 family of stress proteins complexed with antigenic peptides. Incertain embodiments, HSPPCs comprise hsc70, hsp70, hsp90, hsp110,grp170, gp96, calreticulin, or combinations of two or more thereof.

In a specific embodiment, an anti-CTLA-4 antibody described herein isadministered to a subject in combination with a heat shock proteinpeptide complex (HSPPC), e.g., heat shock protein peptide complex-96(HSPPC-96), to treat cancer. HSPPC-96 comprises a 96 kDa heat shockprotein (Hsp), gp96, complexed to antigenic peptides. HSPPC-96 is acancer immunotherapy manufactured from a subject's tumor and containsthe cancer's antigenic “fingerprint.” In certain embodiments, thisfingerprint contains unique antigens that are present only in thatparticular subject's specific cancer cells and injection of the vaccineis intended to stimulate the subject's immune system to recognize andattack any cells with the specific cancer fingerprint.

In certain embodiments, the HSPPC, e.g., HSPPC-96, is produced from thetumor tissue of a subject. In a specific embodiment, the HSPPC (e.g.,HSPPC-96) is produced from a tumor of the type of cancer or metastasisthereof being treated. In another specific embodiment, the HSPPC (e.g.,HSPPC-96) is autologous to the subject being treated. In certainembodiments, the tumor tissue is non-necrotic tumor tissue. In certainembodiments, at least 1 gram (e.g., at least 1, at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,or at least 10 grams) of non-necrotic tumor tissue is used to produce avaccine regimen. In certain embodiments, after surgical resection,non-necrotic tumor tissue is frozen prior to use in vaccine preparation.In some embodiments, the HSPPC, e.g., HSPPC-96, is isolated from thetumor tissue by purification techniques, filtered and prepared for aninjectable vaccine. In certain embodiments, a subject is administered6-12 doses of the HSPPC, e.g., HSPCC-96. In such embodiments, the HSPPC,e.g., HSPPC-96, doses may be administered weekly for the first 4 dosesand then biweekly for the 2-8 additional doses.

Further examples of HSPPCs that may be used in accordance with themethods described herein are disclosed in the following patents andpatent applications, which are incorporated herein by reference hereinin their entireties, U.S. Pat. Nos. 6,391,306, 6,383,492, 6,403,095,6,410,026, 6,436,404, 6,447,780, 6,447,781 and 6,610,659, all of whichare herein incorporated by reference in their entireties.

In certain embodiments, the present invention relates to (a) ananti-CTLA-4 antibody of the invention and/or a pharmaceuticalcomposition of the invention comprising an anti-CTLA-4 antibody of theinvention and a pharmaceutically acceptable carrier or excipient and (b)a vaccine, for use as a medicament. In a preferred embodiment, thevaccine is a heat shock protein based tumor vaccine or a heat shockprotein based pathogen vaccine. In a preferred embodiment, the vaccineis a heat shock protein based viral vaccine.

In certain embodiments, the present invention relates to (a) ananti-CTLA-4 antibody of the invention and/or a pharmaceuticalcomposition of the invention comprising an anti-CTLA-4 antibody of theinvention and a pharmaceutically acceptable carrier or excipient and (b)a vaccine, for use in a method for the treatment of cancer. In apreferred embodiment, the vaccine is a heat shock protein based tumorvaccine.

The anti-CTLA-4 antibody and the additional therapeutic agent (e.g.,chemotherapeutic, checkpoint targeting agent, IDO inhibitor, and/orvaccine) can be administered separately, sequentially or concurrently asseparate dosage forms. In one embodiment, an anti-CTLA-4 antibody isadministered parenterally, and an IDO inhibitor is administered orally.

In certain embodiments, an anti-CTLA-4 antibody described herein isadministered to a subject intratumorally. In certain embodiments, ananti-CTLA-4 antibody described herein is administered to a subjectintratumorally in combination with an additional therapeutic agent. Incertain embodiments, the additional therapeutic agent is administeredsystemically. In certain embodiments, the subject has solid tumors. Incertain embodiments, the subject has head and neck squamous cellcarcinoma (HNSCC). In certain embodiments, the subject has HER2′ breastcancer. In certain embodiments, the additional therapeutic agent that isadministered systemically is an anti-PD-1 antibody (e.g., pembrolizumabor nivolumab). In certain embodiments, the additional therapeutic agentthat is administered systemically is an anti-EGFR antibody (e.g.,cetuximab). In certain embodiments, the additional therapeutic agentthat is administered systemically is an anti-HER2 antibody (e.g.,trastuzumab). In certain embodiments, the additional therapeutic agentthat is administered systemically is a chemotherapeutic agent (e.g.,gemcitabine). In certain embodiments, the subject has solid tumors andthe additional therapeutic agent that is administered systemically is ananti-PD-1 antibody (e.g., pembrolizumab or nivolumab). In certainembodiments, the anti-PD-1 antibody is pembrolizumab administered at 200mg every three weeks. In certain embodiments, the subject has head andneck squamous cell carcinoma (HNSCC) and the additional therapeuticagent that is administered systemically is an anti-EGFR antibody (e.g.,cetuximab). In certain embodiments, the subject has HER2′ breast cancerand the additional therapeutic agent that is administered systemicallyis an anti-HER2 antibody (e.g., trastuzumab). In certain embodiments,the subject further received a chemotherapeutic agent (e.g.,gemcitabine). In one aspect, the present invention relates to ananti-CTLA-4 antibody and/or pharmaceutical composition of the presentinvention, and optionally an additional therapeutic agent, for use in amethod for the treatment of cancer, wherein the anti-CTLA-4 antibodyand/or pharmaceutical composition of the present invention isadministered intratumorally to the subject. In one preferred embodiment,an additional therapeutic agent is administered to the subject, morepreferably, an additional therapeutic agent is administered systemicallyto the subject.

In certain embodiments, an anti-PD-1 antibody is used in methodsdescribed herein. In certain embodiments, the anti-PD-1 antibody isNivolumab, also known as BMS-936558 or MDX1106, developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-1 antibody isPembrolizumab, also known as Lambrolizumab or MK-3475, developed byMerck & Co. In certain embodiments, the anti-PD-1 antibody isPidilizumab, also known as CT-011, developed by CureTech. In certainembodiments, the anti-PD-1 antibody is MEDI0680, also known as AMP-514,developed by Medimmune. In certain embodiments, the anti-PD-1 antibodyis PDR001 developed by Novartis Pharmaceuticals. In certain embodiments,the anti-PD-1 antibody is REGN2810 developed by RegeneronPharmaceuticals. In certain embodiments, the anti-PD-1 antibody isPF-06801591 developed by Pfizer. In certain embodiments, the anti-PD-1antibody is BGB-A317 developed by BeiGene. In certain embodiments, theanti-PD-1 antibody is TSR-042 developed by AnaptysBio and Tesaro. Incertain embodiments, the anti-PD-1 antibody is SHR-1210 developed byHengrui.

Further non-limiting examples of anti-PD-1 antibodies that may be usedin treatment methods described herein are disclosed in the followingpatents and patent applications, which are incorporated herein byreference in their entireties for all purposes: U.S. Pat. No. 6,808,710;U.S. Pat. No. 7,332,582; U.S. Pat. No. 7,488,802; U.S. Pat. No.8,008,449; U.S. Pat. No. 8,114,845; U.S. Pat. No. 8,168,757; U.S. Pat.No. 8,354,509; U.S. Pat. No. 8,686,119; U.S. Pat. No. 8,735,553; U.S.Pat. No. 8,747,847; U.S. Pat. No. 8,779,105; U.S. Pat. No. 8,927,697;U.S. Pat. No. 8,993,731; U.S. Pat. No. 9,102,727; U.S. Pat. No.9,205,148; U.S. Publication No. US 2013/0202623 A1; U.S. Publication No.US 2013/0291136 A1; U.S. Publication No. US 2014/0044738 A1; U.S.Publication No. US 2014/0356363 A1; U.S. Publication No. US 2016/0075783A1; and PCT Publication No. WO 2013/033091 A1; PCT Publication No. WO2015/036394 A1; PCT Publication No. WO 2014/179664 A2; PCT PublicationNo. WO 2014/209804 A1; PCT Publication No. WO 2014/206107 A1; PCTPublication No. WO 2015/058573 A1; PCT Publication No. WO 2015/085847A1; PCT Publication No. WO 2015/200119 A1; PCT Publication No. WO2016/015685 A1; and PCT Publication No. WO 2016/020856 A1.

In certain embodiments, an anti-PD-L1 antibody is used in methodsdescribed herein. In certain embodiments, the anti-PD-L1 antibody isatezolizumab developed by Genentech. In certain embodiments, theanti-PD-L1 antibody is durvalumab developed by AstraZeneca, Celgene andMedimmune. In certain embodiments, the anti-PD-L1 antibody is avelumab,also known as MSB0010718C, developed by Merck Serono and Pfizer. Incertain embodiments, the anti-PD-L1 antibody is MDX-1105 developed byBristol-Myers Squibb. In certain embodiments, the anti-PD-L1 antibody isAMP-224 developed by Amplimmune and GSK.

Non-limiting examples of anti-PD-L1 antibodies that may be used intreatment methods described herein are disclosed in the followingpatents and patent applications, which are incorporated herein byreference in their entireties for all purposes: U.S. Pat. No. 7,943,743;U.S. Pat. No. 8,168,179; U.S. Pat. No. 8,217,149; U.S. Pat. No.8,552,154; U.S. Pat. No. 8,779,108; U.S. Pat. No. 8,981,063; U.S. Pat.No. 9,175,082; U.S. Publication No. US 2010/0203056 A1; U.S. PublicationNo. US 2003/0232323 A1; U.S. Publication No. US 2013/0323249 A1; U.S.Publication No. US 2014/0341917 A1; U.S. Publication No. US 2014/0044738A1; U.S. Publication No. US 2015/0203580 A1; U.S. Publication No. US2015/0225483 A1; U.S. Publication No. US 2015/0346208 A1; U.S.Publication No. US 2015/0355184 A1; and PCT Publication No. WO2014/100079 A1; PCT Publication No. WO 2014/022758 A1; PCT PublicationNo. WO 2014/055897 A2; PCT Publication No. WO 2015/061668 A1; PCTPublication No. WO 2015/109124 A1; PCT Publication No. WO 2015/195163A1; PCT Publication No. WO 2016/000619 A1; and PCT Publication No. WO2016/030350 A1.

In certain embodiments, an anti-LAG-3 antibody is used in methodsdescribed herein. In certain embodiments, the anti-LAG-3 antibody isBMS-986016 developed by Bristol-Myers Squibb. In certain embodiments,the anti-LAG-3 antibody is LAG525 developed by Novartis. In certainembodiments, the anti-LAG-3 antibody is GSK2831781 developed by GSK.

Non-limiting examples of anti-LAG-3 antibodies that may be used intreatment methods described herein are disclosed in the followingpatents and patent applications, which are incorporated herein byreference in their entireties for all purposes: U.S. Pat. No. 9,244,059;U.S. Publication No. US 2011/0150892 A1; U.S. Publication No. US2014/0093511 A1; U.S. Publication No. US 2014/0286935 A1; U.S.Publication No. US 2015/0259420 A1; and PCT Publication No. WO2015/042246 A1; PCT Publication No. WO 2015/116539 A1; PCT PublicationNo. WO 2015/200119 A1; and PCT Publication No. WO 2016/028672 A1.

In certain embodiments, an anti-EGFR antibody is used in methodsdescribed herein. In certain embodiments, the anti-EGFR antibody iscetuximab developed by Bristol-Myers Squibb and ImClone, panitumumabdeveloped by Abgenix and Amgen, nimotuzumab developed by CMI Cuba and YMBioSciences, necitumumab developed by ImClone, zalutumumab developed byGenmab, matuzumab developed by Takeda, Sym004 developed by Merck Seronoand Symphogen, imgatuzumab developed by Glycart and Roche, duligotumabdeveloped by Genentech and Roche, depatuxizumab developed by Abbott,depatuxizumab mafodotin developed by Abbvie, MM-151 developed by Adimaband Merrimack, GC1118 developed by Green Cross, AMG 595 developed byAmgen and ImmunoGen, CetuGEX developed by Glycotope, laprituximabemtansine developed by ImmunoGen, JNJ-61186372 developed by Genmab andJanssen Biotech, SCT200 developed by Sinocelltech, LY3164530 developedby Lilly, HLX07 developed by Shanghai Henlius, or SYN004 developed bySynermore.

In certain embodiments, an anti-HER2 antibody is used in methodsdescribed herein. In certain embodiments, the anti-HER2 antibody istrastuzumab developed by Genentech and Roche, trastuzumab emtansinedeveloped by Genentech and Roche, pertuzumab developed by Genentech,ertumaxomab developed by Fresenius, margetuximab developed byMacroGenics, MM-111 developed by Merrimack, CT-P06 developed byCelltrion, PF-05280014 developed by Pfizer, MM-302 developed byMerrimack, SB3 developed by Merck & Co, CMAB302 developed by Shanghai CPGuojian, TrasGEX developed by Glycotope, ARX788 developed by Ambrx andZhejiang Medicine, SYD985 developed by Synthon, FS102 developed byBristol-Myers Squibb and f-star, BCD-022 developed by Biocad, ABP 980developed by Amgen, DS-8201a developed by Daiichi Sankyo, HLX02developed by Shanghai Henlius, or CANMAb developed by Biocon and Mylan.

An antibody or pharmaceutical composition described herein may bedelivered to a subject by a variety of routes. These include, but arenot limited to, parenteral, intranasal, intratracheal, oral,intradermal, topical, intramuscular, intraperitoneal, transdermal,intravenous, intratumoral, conjunctival and subcutaneous routes.Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent for useas a spray. In certain embodiments, the antibody or pharmaceuticalcomposition described herein is delivered subcutaneously orintravenously. In certain embodiments, the antibody or pharmaceuticalcomposition described herein is delivered intratumorally. In certainembodiments, the anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is delivered to a tumor draining lymph node. In certainembodiments, the antibody or pharmaceutical composition described hereinis delivered via a localized administration (e.g., subcutaneousadministration). In certain embodiments, the anti-CTLA-4 antibody orpharmaceutical composition described herein is delivered systemically.In certain embodiments, the anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is delivered locally.

In one aspect, the present invention relates to an anti-CTLA-4 antibodyand/or pharmaceutical composition of the present invention, andoptionally an additional therapeutic agent, for use in a method for thetreatment of cancer, wherein the anti-CTLA-4 antibody and/orpharmaceutical composition of the present invention is deliveredintratumorally to the subject, is delivered to a tumor draining lymphnode of a subject, or is delivered via a localized administration (e.g.,subcutaneous administration) to a subject.

The amount of an antibody or composition which will be effective in thetreatment and/or prevention of a condition will depend on the nature ofthe disease, and can be determined by standard clinical techniques.

The precise dose to be employed in a composition will also depend on theroute of administration, and the seriousness of the infection or diseasecaused by it, and should be decided according to the judgment of thepractitioner and each subject's circumstances. For example, effectivedoses may also vary depending upon means of administration, target site,physiological state of the patient (including age, body weight andhealth), whether the patient is human or an animal, other medicationsadministered, or whether treatment is prophylactic or therapeutic.Usually, the patient is a human but non-human mammals includingtransgenic mammals can also be treated. Treatment dosages are optimallytitrated to optimize safety and efficacy.

In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg,1 mg/kg, 3 mg/kg, 6 mg/kg, 10 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg,about 1 mg/kg, about 3 mg/kg, about 6 mg/kg, or about 10 mg/kg. Incertain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) every week, every two weeks, every three weeks,every four weeks, every six weeks, every eight weeks, every twelveweeks, every month, every two months, every three months, every fourmonths, every five months, every six months, every eight months, andevery year, e.g., at the doses described above. In certain embodiments,an anti-CTLA-4 antibody or pharmaceutical composition described hereinis administered to a subject (e.g., via intravenous injection) everythree weeks at the doses described above.

In one aspect, the present invention relates to an anti-CTLA-4 antibodyand/or pharmaceutical composition of the present invention, andoptionally an additional therapeutic agent, for use in a method for thetreatment of cancer, wherein the anti-CTLA-4 antibody and/orpharmaceutical composition of the present invention is administered to asubject at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3mg/kg, 6 mg/kg, 10 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1mg/kg, about 3 mg/kg, about 6 mg/kg, or about 10 mg/kg, more preferablyevery two weeks, every three weeks, every four weeks, every six weeks,or every twelve weeks.

In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) at 0.1 mg/kg every two weeks, every three weeks,every four weeks, every six weeks, or every twelve weeks. In certainembodiments, an anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered to a subject (e.g., via intravenousinjection) at 0.3 mg/kg every two weeks, every three weeks, every fourweeks, every six weeks, or every twelve weeks. In certain embodiments,an anti-CTLA-4 antibody or pharmaceutical composition described hereinis administered to a subject (e.g., via intravenous injection) at 1mg/kg every two weeks, every three weeks, every four weeks, every sixweeks, or every twelve weeks. In certain embodiments, an anti-CTLA-4antibody or pharmaceutical composition described herein is administeredto a subject (e.g., via intravenous injection) at 3 mg/kg every twoweeks, every three weeks, every four weeks, every six weeks, or everytwelve weeks. In certain embodiments, an anti-CTLA-4 antibody orpharmaceutical composition described herein is administered to a subject(e.g., via intravenous injection) at 6 mg/kg every two weeks, everythree weeks, every four weeks, every six weeks, or every twelve weeks.In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) at 10 mg/kg every two weeks, every three weeks,every four weeks, every six weeks, or every twelve weeks.

In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) at 0.1 mg/kg or about 0.1 mg/kg every threeweeks. In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) at 0.3 mg/kg or about 0.3 mg/kg every threeweeks. In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) at 1 mg/kg or about 1 mg/kg every three weeks. Incertain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) at 3 mg/kg or about 3 mg/kg every three weeks. Incertain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) at 6 mg/kg or about 6 mg/kg every three weeks. Incertain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject (e.g., viaintravenous injection) at 10 mg/kg or about 10 mg/kg every three weeks.

In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject viaintratumoral injection at 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg,1 mg/kg, 3 mg/kg, about 0.01 mg/kg, about 0.03 mg/kg, about 0.1 mg/kg,about 0.3 mg/kg, about 1 mg/kg, or about 3 mg/kg. In certainembodiments, an anti-CTLA-4 antibody or pharmaceutical compositiondescribed herein is administered to a subject via intratumoral injectionevery week, every two weeks, every three weeks, every four weeks, everysix weeks, every eight weeks, every twelve weeks, every month, every twomonths, every three months, every four months, every five months, everysix months, every eight months, and every year, e.g., at the dosesdescribed above. In certain embodiments, an anti-CTLA-4 antibody orpharmaceutical composition described herein is administered to a subjectvia intratumoral injection every three weeks at the doses describedabove.

In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject viaintratumoral injection at 0.01 mg/kg or about 0.01 mg/kg every threeweeks. In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject viaintratumoral injection at 0.03 mg/kg or about 0.03 mg/kg every threeweeks. In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject viaintratumoral injection at 0.1 mg/kg or about 0.1 mg/kg every threeweeks. In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject viaintratumoral injection at 0.3 mg/kg or about 0.3 mg/kg every threeweeks. In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject viaintratumoral injection at 1 mg/kg or about 1 mg/kg every three weeks. Incertain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject viaintratumoral injection at 3 mg/kg or about 3 mg/kg every three weeks.

In certain embodiments, an anti-CTLA-4 antibody or pharmaceuticalcomposition described herein is administered to a subject via alocalized administration (e.g., subcutaneous administration) at 0.01mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, about 0.01mg/kg, about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1mg/kg, or about 3 mg/kg. In certain embodiments, an anti-CTLA-4 antibodyor pharmaceutical composition described herein is administered to asubject via a localized administration (e.g., subcutaneousadministration) every week, every two weeks, every three weeks, everyfour weeks, every six weeks, every eight weeks, every twelve weeks,every month, every two months, every three months, every four months,every five months, every six months, every eight months, and every year,e.g., at the doses described above.

In certain embodiments, the therapeutic combination is administered to asubject for at least 3, 6, 9, 12, 18, or 24 months. In certainembodiments, the therapeutic combination is administered to a subjectfor up to 3, 6, 9, 12, 18, or 24 months.

In certain embodiments, the instant disclosure provides a method oftreating a subject having angiosarcoma, the method comprisingadministering to the subject (e.g., intravenously, intratumorally, orsubcutaneously) an effective amount of an anti-CTLA-4 antibody orpharmaceutical composition described herein. In certain embodiments, theinstant disclosure provides a method of treating a subject havingangiosarcoma, the method comprising administering to the subjectintravenously an antibody that specifically binds to human CTLA-4 at0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg, 0.3 mg/kg, 1 mg/kg, 3 mg/kg, about0.01 mg/kg, about 0.03 mg/kg, about 0.1 mg/kg, about 0.3 mg/kg, about 1mg/kg, or about 3 mg/kg, optionally every one, two or three weeks. Incertain embodiments, the instant disclosure provides a method oftreating a subject having angiosarcoma, the method comprisingadministering to the subject intravenously an antibody that specificallybinds to human CTLA-4 at 0.1 mg/kg once every three weeks. In certainembodiments, the antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 8 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 9. Incertain embodiments, the antibody comprises a heavy chain comprising theamino acid sequence of SEQ ID NO: 23; and a light chain comprising theamino acid sequence of SEQ ID NO: 27. In certain embodiments, theantibody comprises a heavy chain comprising the amino acid sequence ofSEQ ID NO: 24; and a light chain comprising the amino acid sequence ofSEQ ID NO: 27. In certain embodiments, the antibody comprises a heavychain comprising the amino acid sequence of SEQ ID NO: 25; and a lightchain comprising the amino acid sequence of SEQ ID NO: 27. In certainembodiments, the antibody comprises a heavy chain comprising the aminoacid sequence of SEQ ID NO: 26; and a light chain comprising the aminoacid sequence of SEQ ID NO: 27.

An anti-CTLA-4 antibody described herein can also be used to assayCTLA-4 protein levels in a biological sample using classicalimmunohistological methods known to those of skill in the art, includingimmunoassays, such as the enzyme linked immunosorbent assay (ELISA),immunoprecipitation, or Western blotting. Suitable antibody assay labelsare known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C),sulfur (³⁵S), tritium (³H), indium (¹²¹In), and technetium (⁹⁹Tc);luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin. Such labels can be used to labelan antibody or an antigen-binding fragment thereof described herein.Alternatively, a second antibody that recognizes an anti-CTLA-4 antibodyor antigen-binding fragment thereof described herein can be labeled andused in combination with an anti-CTLA-4 antibody or antigen-bindingfragment thereof to detect CTLA-4 protein levels. In one embodiment, thepresent invention relates to the use of an anti-CTLA-4 antibody of theinvention, for assaying and/or detecting CTLA-4 protein levels in abiological sample in vitro.

Assaying for the expression level of CTLA-4 protein is intended toinclude qualitatively or quantitatively measuring or estimating thelevel of a CTLA-4 protein in a first biological sample either directly(e.g., by determining or estimating absolute protein level) orrelatively (e.g., by comparing to the disease associated protein levelin a second biological sample). CTLA-4 polypeptide expression level inthe first biological sample can be measured or estimated and compared toa standard CTLA-4 protein level, the standard being taken from a secondbiological sample obtained from an individual not having the disorder orbeing determined by averaging levels from a population of individualsnot having the disorder. As will be appreciated in the art, once the“standard” CTLA-4 polypeptide level is known, it can be used repeatedlyas a standard for comparison.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing CTLA-4. Methods for obtaining tissuebiopsies and body fluids from animals (e.g., humans) are well known inthe art. Biological samples include peripheral mononuclear blood cells.

An anti-CTLA-4 antibody or antigen-binding fragment thereof describedherein can be used for prognostic, diagnostic, monitoring and screeningapplications, including in vitro and in vivo applications well known andstandard to the skilled artisan and based on the present description.Prognostic, diagnostic, monitoring and screening assays and kits for invitro assessment and evaluation of immune system status and/or immuneresponse may be utilized to predict, diagnose and monitor to evaluatepatient samples including those known to have or suspected of having animmune system-dysfunction or with regard to an anticipated or desiredimmune system response, antigen response or vaccine response. Theassessment and evaluation of immune system status and/or immune responseis also useful in determining the suitability of a patient for aclinical trial of a drug or for the administration of a particularchemotherapeutic agent or an antibody or antigen-binding fragmentthereof, including combinations thereof, versus a different agent orantibody or antigen-binding fragment thereof. This type of prognosticand diagnostic monitoring and assessment is already in practiceutilizing antibodies against the HER2 protein in breast cancer(HercepTest™, Dako) where the assay is also used to evaluate patientsfor antibody therapy using Herceptin®. In vivo applications includedirected cell therapy and immune system modulation and radio imaging ofimmune responses.

In one aspect, the present invention relates to an anti-CTLA-4 antibodyand/or pharmaceutical composition of the present invention for use as adiagnostic.

In one aspect, the present invention relates to an anti-CTLA-4 antibodyand/or pharmaceutical composition of the present invention for use in amethod for the prediction, diagnosis and/or monitoring of an immunesystem-dysfunction and/or cancer.

In one embodiment, the present invention relates to the use of ananti-CTLA-4 antibody of the invention, for predicting, diagnosing and/ormonitoring an immune system-dysfunction and/or cancer in a subject byassaying and/or detecting CTLA-4 protein levels in a biological sampleof the subject of in vitro.

In one embodiment, an anti-CTLA-4 antibody or antigen-binding fragmentthereof can be used in immunohistochemistry of biopsy samples. Inanother embodiment, an anti-CTLA-4 antibody or antigen-binding fragmentthereof can be used to detect levels of CTLA-4, or levels of cells whichcontain CTLA-4 on their membrane surface, which levels can then belinked to certain disease symptoms. Anti-CTLA-4 antibodies orantigen-binding fragments thereof described herein may carry adetectable or functional label. When fluorescence labels are used,currently available microscopy and fluorescence-activated cell sorteranalysis (FACS) or combination of both methods procedures known in theart may be utilized to identify and to quantitate the specific bindingmembers. Anti-CTLA-4 antibodies or antigen-binding fragments thereofdescribed herein may carry a fluorescence label. Exemplary fluorescencelabels include, for example, reactive and conjugated probes e.g.Aminocoumarin, Fluorescein and Texas red, Alexa Fluor dyes, Cy dyes andDyLight dyes. An anti-CTLA-4 antibody or antigen-binding fragmentthereof may carry a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹¹⁷Lu,¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Ac and ¹⁸⁶Re. Whenradioactive labels are used, currently available counting proceduresknown in the art may be utilized to identify and quantitate the specificbinding of anti-CTLA-4 antibody or antigen-binding fragment thereof toCTLA-4 (e.g., human CTLA-4). In the instance where the label is anenzyme, detection may be accomplished by any of the presently utilizedcolorimetric, spectrophotometric, fluoro spectrophotometric,amperometric or gasometric techniques as known in the art. This can beachieved by contacting a sample or a control sample with an anti-CTLA-4antibody or antigen-binding fragment thereof under conditions that allowfor the formation of a complex between the antibody or antigen-bindingfragment thereof and CTLA-4. Any complexes formed between the antibodyor antigen-binding fragment thereof and CTLA-4 are detected and comparedin the sample and the control. In light of the specific binding of theantibodies described herein for CTLA-4, the antibodies orantigen-binding fragments thereof can be used to specifically detectCTLA-4 expression on the surface of cells. The antibodies orantigen-binding fragments thereof described herein can also be used topurify CTLA-4 via immunoaffinity purification. Also included herein isan assay system which may be prepared in the form of a test kit for thequantitative analysis of the extent of the presence of, for instance,CTLA-4 or CTLA-4/CTLA-4 ligand complexes. The system or test kit maycomprise a labeled component, e.g., a labeled antibody, and one or moreadditional immunochemical reagents.

In one embodiment, the present invention relates to an in vitro methodfor assaying and/or detecting CTLA-4 protein levels in a biologicalsample comprising (1) contacting a sample and optionally a controlsample with an anti-CTLA-4 antibody or antigen-binding fragment thereofof the invention under conditions that allow for the formation of acomplex between the antibody or antigen-binding fragment thereof andCTLA-4, and (2) detecting and comparing the complexes formed in thesample and optionally the control.

6.5 Polynucleotides, Vectors and Methods of Producing Anti-CTLA-4Antibodies

In another aspect, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody described herein or a fragmentthereof (e.g., a light chain variable region and/or heavy chain variableregion) that specifically binds to a CTLA-4 (e.g., human CTLA-4)antigen, and vectors, e.g., vectors comprising such polynucleotides forrecombinant expression in host cells (e.g., E. coli and mammaliancells). Provided herein are polynucleotides comprising nucleotidesequences encoding any of the antibodies provided herein, as well asvectors comprising such polynucleotide sequences, e.g., expressionvectors for their efficient expression in host cells, e.g., mammaliancells.

As used herein, an “isolated” polynucleotide or nucleic acid molecule isone which is separated from other nucleic acid molecules which arepresent in the natural source (e.g., in a mouse or a human) of thenucleic acid molecule. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized. For example, the language “substantially free”includes preparations of polynucleotide or nucleic acid molecule havingless than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (in particular lessthan about 10%) of other material, e.g., cellular material, culturemedium, other nucleic acid molecules, chemical precursors and/or otherchemicals. In a specific embodiment, a nucleic acid molecule(s) encodingan antibody described herein is isolated or purified.

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies or antigen-binding fragmentsthereof, which specifically bind to a CTLA-4 polypeptide (e.g., humanCTLA-4) and comprises an amino acid sequence as described herein, aswell as antibodies which compete with such antibodies for binding to aCTLA-4 polypeptide (e.g., in a dose-dependent manner), or which binds tothe same epitope as that of such antibodies.

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding the light chain or heavy chain of anantibody described herein. The polynucleotides can comprise nucleotidesequences encoding a light chain comprising the VL FRs and CDRs ofantibodies described herein (see, e.g., Table 1).

Also provided herein are polynucleotides encoding an anti-CTLA-4antibody that are optimized, e.g., by codon/RNA optimization,replacement with heterologous signal sequences, and elimination of mRNAinstability elements. Methods to generate optimized nucleic acidsencoding an anti-CTLA-4 antibody or a fragment thereof (e.g., lightchain, heavy chain, VH domain, or VL domain) for recombinant expressionby introducing codon changes and/or eliminating inhibitory regions inthe mRNA can be carried out by adapting the optimization methodsdescribed in, e.g., U.S. Pat. Nos. 5,965,726; 6,174,666; 6,291,664;6,414,132; and 6,794,498, accordingly, all of which are hereinincorporated by reference in their entireties. For example, potentialsplice sites and instability elements (e.g., A/T or A/U rich elements)within the RNA can be mutated without altering the amino acids encodedby the nucleic acid sequences to increase stability of the RNA forrecombinant expression. The alterations utilize the degeneracy of thegenetic code, e.g., using an alternative codon for an identical aminoacid. In some embodiments, it can be desirable to alter one or morecodons to encode a conservative mutation, e.g., a similar amino acidwith similar chemical structure and properties and/or function as theoriginal amino acid. Such methods can increase expression of ananti-CTLA-4 antibody or fragment thereof by at least 1 fold, 2 fold, 3fold, 4 fold, 5 fold, 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60fold, 70 fold, 80 fold, 90 fold, or 100 fold or more relative to theexpression of an anti-CTLA-4 antibody encoded by polynucleotides thathave not been optimized.

In certain embodiments, an optimized polynucleotide sequence encoding ananti-CTLA-4 antibody described herein or a fragment thereof (e.g., VLdomain and/or VH domain) can hybridize to an antisense (e.g.,complementary) polynucleotide of an unoptimized polynucleotide sequenceencoding an anti-CTLA-4 antibody described herein or a fragment thereof(e.g., VL domain and/or VH domain). In specific embodiments, anoptimized nucleotide sequence encoding an anti-CTLA-4 antibody describedherein or a fragment hybridizes under high stringency conditions toantisense polynucleotide of an unoptimized polynucleotide sequenceencoding an anti-CTLA-4 antibody described herein or a fragment thereof.In a specific embodiment, an optimized nucleotide sequence encoding ananti-CTLA-4 antibody described herein or a fragment thereof hybridizesunder high stringency, intermediate or lower stringency hybridizationconditions to an antisense polynucleotide of an unoptimized nucleotidesequence encoding an anti-CTLA-4 antibody described herein or a fragmentthereof. Information regarding hybridization conditions has beendescribed, see, e.g., U.S. Patent Application Publication No. US2005/0048549 (e.g., paragraphs 72-73), which is incorporated herein byreference.

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. Nucleotidesequences encoding antibodies described herein, e.g., antibodiesdescribed in Table 1, and modified versions of these antibodies can bedetermined using methods well known in the art, i.e., nucleotide codonsknown to encode particular amino acids are assembled in such a way togenerate a nucleic acid that encodes the antibody. Such a polynucleotideencoding the antibody can be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier G et al., (1994),BioTechniques 17: 242-6, incorporated by reference in its entirety),which, briefly, involves the synthesis of overlapping oligonucleotidescontaining portions of the sequence encoding the antibody, annealing andligating of those oligonucleotides, and then amplification of theligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody described hereincan be generated from nucleic acid from a suitable source (e.g., ahybridoma) using methods well known in the art (e.g., PCR and othermolecular cloning methods). For example, PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of a known sequencecan be performed using genomic DNA obtained from hybridoma cellsproducing the antibody of interest. Such PCR amplification methods canbe used to obtain nucleic acids comprising the sequence encoding thelight chain and/or heavy chain of an antibody. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody. The amplified nucleic acids can be cloned intovectors for expression in host cells and for further cloning, forexample, to generate chimeric and humanized antibodies.

If a clone containing a nucleic acid encoding a particular antibody isnot available, but the sequence of the antibody molecule is known, anucleic acid encoding the immunoglobulin can be chemically synthesizedor obtained from a suitable source (e.g., an antibody cDNA library or acDNA library generated from, or nucleic acid, preferably poly A+ RNA,isolated from, any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody described herein) by PCRamplification using synthetic primers hybridizable to the 3′ and 5′ endsof the sequence or by cloning using an oligonucleotide probe specificfor the particular gene sequence to identify, e.g., a cDNA clone from acDNA library that encodes the antibody. Amplified nucleic acidsgenerated by PCR can then be cloned into replicable cloning vectorsusing any method well known in the art.

DNA encoding anti-CTLA-4 antibodies described herein can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of the anti-CTLA-4 antibodies).Hybridoma cells can serve as a source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as E. coli cells, simian COS cells, Chinese hamsterovary (CHO) cells (e.g., CHO cells from the CHO GS System™ (Lonza)), ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of anti-CTLA-4 antibodies in the recombinant hostcells.

To generate whole antibodies, PCR primers including VH or VL nucleotidesequences, a restriction site, and a flanking sequence to protect therestriction site can be used to amplify the VH or VL sequences in scFvclones. Utilizing cloning techniques known to those of skill in the art,the PCR amplified VH domains can be cloned into vectors expressing aheavy chain constant region, e.g., the human gamma 4 constant region,and the PCR amplified VL domains can be cloned into vectors expressing alight chain constant region, e.g., human kappa or lambda constantregions. In certain embodiments, the vectors for expressing the VH or VLdomains comprise an EF-1α promoter, a secretion signal, a cloning sitefor the variable region, constant domains, and a selection marker suchas neomycin. The VH and VL domains can also be cloned into one vectorexpressing the necessary constant regions. The heavy chain conversionvectors and light chain conversion vectors are then co-transfected intocell lines to generate stable or transient cell lines that expressfull-length antibodies, e.g., IgG, using techniques known to those ofskill in the art.

The DNA also can be modified, for example, by substituting the codingsequence for human heavy and light chain constant domains in place ofthe murine sequences, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide.

Also provided are polynucleotides that hybridize under high stringency,intermediate or lower stringency hybridization conditions topolynucleotides that encode an antibody described herein. In specificembodiments, polynucleotides described herein hybridize under highstringency, intermediate or lower stringency hybridization conditions topolynucleotides encoding a VH domain and/or VL domain provided herein.

Hybridization conditions have been described in the art and are known toone of skill in the art. For example, hybridization under stringentconditions can involve hybridization to filter-bound DNA in 6× sodiumchloride/sodium citrate (SSC) at about 45° C. followed by one or morewashes in 0.2×SSC/0.1% SDS at about 50-65° C.; hybridization underhighly stringent conditions can involve hybridization to filter-boundnucleic acid in 6×SSC at about 45° C. followed by one or more washes in0.1×SSC/0.2% SDS at about 68° C. Hybridization under other stringenthybridization conditions are known to those of skill in the art and havebeen described, see, for example, Ausubel F M et al., eds., (1989)Current Protocols in Molecular Biology, Vol. I, Green PublishingAssociates, Inc. and John Wiley & Sons, Inc., New York at pages6.3.1-6.3.6 and 2.10.3, incorporated by reference in its entirety.

In certain aspects, provided herein are cells (e.g., host cells)expressing (e.g., recombinantly) antibodies described herein (or anantigen-binding fragment thereof) which specifically bind to CTLA-4(e.g., human CTLA-4) and related polynucleotides and expression vectors.Provided herein are vectors (e.g., expression vectors) comprisingpolynucleotides comprising nucleotide sequences encoding anti-CTLA-4antibodies or a fragment for recombinant expression in host cells,preferably in mammalian cells. Also provided herein are host cellscomprising such vectors for recombinantly expressing anti-CTLA-4antibodies described herein (e.g., human or humanized antibody). In aparticular aspect, provided herein are methods for producing an antibodydescribed herein, comprising expressing such antibody from a host cell.

Recombinant expression of an antibody described herein (e.g., afull-length antibody, heavy and/or light chain of an antibody, or asingle chain antibody described herein) that specifically binds toCTLA-4 (e.g., human CTLA-4) involves construction of an expressionvector containing a polynucleotide that encodes the antibody. Once apolynucleotide encoding an antibody molecule, heavy and/or light chainof an antibody, or a fragment thereof (e.g., heavy and/or light chainvariable regions) described herein has been obtained, the vector for theproduction of the antibody molecule can be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody or antibody fragment (e.g., light chain or heavy chain)encoding nucleotide sequence are described herein. Methods which arewell known to those skilled in the art can be used to constructexpression vectors containing antibody or antibody fragment (e.g., lightchain or heavy chain) coding sequences and appropriate transcriptionaland translational control signals. These methods include, for example,in vitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Also provided are replicable vectors comprising anucleotide sequence encoding an antibody molecule described herein, aheavy or light chain of an antibody, a heavy or light chain variableregion of an antibody or a fragment thereof, or a heavy or light chainCDR, operably linked to a promoter. Such vectors can, for example,include the nucleotide sequence encoding the constant region of theantibody molecule (see, e.g., International Publication Nos. WO 86/05807and WO 89/01036; and U.S. Pat. No. 5,122,464, which are hereinincorporated by reference in their entireties) and variable regions ofthe antibody can be cloned into such a vector for expression of theentire heavy, the entire light chain, or both the entire heavy and lightchains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques and the resulting cells can then be cultured byconventional techniques to produce an antibody described herein or afragment thereof. Thus, provided herein are host cells containing apolynucleotide encoding an antibody described herein or fragmentsthereof, or a heavy or light chain thereof, or fragment thereof, or asingle chain antibody described herein, operably linked to a promoterfor expression of such sequences in the host cell. In certainembodiments, for the expression of double-chained antibodies, vectorsencoding both the heavy and light chains, individually, can beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below. In certain embodiments, ahost cell contains a vector comprising a polynucleotide encoding boththe heavy chain and light chain of an antibody described herein, or afragment thereof. In specific embodiments, a host cell contains twodifferent vectors, a first vector comprising a polynucleotide encoding aheavy chain or a heavy chain variable region of an antibody describedherein, or a fragment thereof, and a second vector comprising apolynucleotide encoding a light chain or a light chain variable regionof an antibody described herein, or a fragment thereof. In otherembodiments, a first host cell comprises a first vector comprising apolynucleotide encoding a heavy chain or a heavy chain variable regionof an antibody described herein, or a fragment thereof, and a secondhost cell comprises a second vector comprising a polynucleotide encodinga light chain or a light chain variable region of an antibody describedherein. In specific embodiments, a heavy chain/heavy chain variableregion expressed by a first cell associated with a light chain/lightchain variable region of a second cell to form an anti-CTLA-4 antibodydescribed herein or an antigen-binding fragment thereof. In certainembodiments, provided herein is a population of host cells comprisingsuch first host cell and such second host cell.

In a particular embodiment, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-CTLA-4 antibody describedherein, and a second vector comprising a polynucleotide encoding a heavychain/heavy chain variable region of an anti-CTLA-4 antibody describedherein.

A variety of host-expression vector systems can be utilized to expressantibody molecules described herein (see, e.g., U.S. Pat. No.5,807,715). Such host-expression systems represent vehicles by which thecoding sequences of interest can be produced and subsequently purified,but also represent cells which can, when transformed or transfected withthe appropriate nucleotide coding sequences, express an antibodymolecule described herein in situ. These include but are not limited tomicroorganisms such as bacteria (e.g., E. coli and B. subtilis)transformed with recombinant bacteriophage DNA, plasmid DNA or cosmidDNA expression vectors containing antibody coding sequences; yeast(e.g., Saccharomyces Pichia) transformed with recombinant yeastexpression vectors containing antibody coding sequences; insect cellsystems infected with recombinant virus expression vectors (e.g.,baculovirus) containing antibody coding sequences; plant cell systems(e.g., green algae such as Chlamydomonas reinhardtii) infected withrecombinant virus expression vectors (e.g., cauliflower mosaic virus,CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors (e.g., Ti plasmid) containing antibody codingsequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS),CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, andNIH 3T3, HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 andBMT10 cells) harboring recombinant expression constructs containingpromoters derived from the genome of mammalian cells (e.g.,metallothionein promoter) or from mammalian viruses (e.g., theadenovirus late promoter; the vaccinia virus 7.5K promoter). In aspecific embodiment, cells for expressing antibodies described herein oran antigen-binding fragment thereof are CHO cells, for example CHO cellsfrom the CHO GS System™ (Lonza). In a particular embodiment, cells forexpressing antibodies described herein are human cells, e.g., human celllines. In a specific embodiment, a mammalian expression vector ispOptiVEC™ or pcDNA3.3. In a particular embodiment, bacterial cells suchas Escherichia coli, or eukaryotic cells (e.g., mammalian cells),especially for the expression of whole recombinant antibody molecule,are used for the expression of a recombinant antibody molecule. Forexample, mammalian cells such as Chinese hamster ovary (CHO) cells, inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies (Foecking M K & Hofstetter H (1986) Gene 45:101-5; and Cockett M I et al., (1990) Biotechnology 8(7): 662-7, whichare herein incorporated by reference in their entireties). In certainembodiments, antibodies described herein are produced by CHO cells orNS0 cells. In a specific embodiment, the expression of nucleotidesequences encoding antibodies described herein which specifically bindCTLA-4 (e.g., human CTLA-4) is regulated by a constitutive promoter,inducible promoter or tissue specific promoter.

In bacterial systems, a number of expression vectors can beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such anantibody is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited to,the E. coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983)EMBO J 2: 1791-1794, herein incorporated by reference in its entirety),in which the antibody coding sequence can be ligated individually intothe vector in frame with the lac Z coding region so that a fusionprotein is produced; pIN vectors (Inouye S & Inouye M (1985) Nuc AcidsRes 13: 3101-3109; Van Heeke G & Schuster S M (1989) J Biol Chem 24:5503-5509, which are herein incorporated by reference in theirentireties); and the like. For example, pGEX vectors can also be used toexpress foreign polypeptides as fusion proteins with glutathione5-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV), for example, can be used as a vector to express foreign genes.The virus grows in Spodoptera frugiperda cells. The antibody codingsequence can be cloned individually into non-essential regions (forexample the polyhedrin gene) of the virus and placed under control of anAcNPV promoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts (e.g., see Logan J &Shenk T (1984) PNAS 81(12): 3655-9, herein incorporated by reference inits entirety). Specific initiation signals can also be required forefficient translation of inserted antibody coding sequences. Thesesignals include the ATG initiation codon and adjacent sequences.Furthermore, the initiation codon must be in phase with the readingframe of the desired coding sequence to ensure translation of the entireinsert. These exogenous translational control signals and initiationcodons can be of a variety of origins, both natural and synthetic. Theefficiency of expression can be enhanced by the inclusion of appropriatetranscription enhancer elements, transcription terminators, etc. (see,e.g., Bitter G et al., (1987) Methods Enzymol. 153: 516-544, hereinincorporated by reference in its entirety).

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, Hela, MDCK, HEK293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NS0 (a murinemyeloma cell line that does not endogenously produce any immunoglobulinchains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst,HEK-293T, HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20, BMT10 andHsS78Bst cells. In certain embodiments, anti-CTLA-4 antibodies describedherein are produced in mammalian cells, such as CHO cells.

In a specific embodiment, the antibodies described herein orantigen-binding fragments thereof have reduced fucose content or nofucose content. Such antibodies can be produced using techniques knownone skilled in the art. For example, the antibodies can be expressed incells deficient or lacking the ability of to fucosylate. In a specificexample, cell lines with a knockout of both alleles ofα1,6-fucosyltransferase can be used to produce antibodies orantigen-binding fragments thereof with reduced fucose content. ThePotelligent® system (Lonza) is an example of such a system that can beused to produce antibodies or antigen-binding fragments thereof withreduced fucose content.

For long-term, high-yield production of recombinant proteins, stableexpression cells can be generated. For example, cell lines which stablyexpress an anti-CTLA-4 antibody described herein or an antigen-bindingfragment thereof can be engineered. In specific embodiments, a cellprovided herein stably expresses a light chain/light chain variableregion and a heavy chain/heavy chain variable region which associate toform an antibody described herein or an antigen-binding fragmentthereof.

In certain aspects, rather than using expression vectors which containviral origins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements (e.g., promoter,enhancer, sequences, transcription terminators, polyadenylation sites,etc.), and a selectable marker. Following the introduction of theforeign DNA/polynucleotide, engineered cells can be allowed to grow for1-2 days in an enriched media, and then are switched to a selectivemedia. The selectable marker in the recombinant plasmid confersresistance to the selection and allows cells to stably integrate theplasmid into their chromosomes and grow to form foci which in turn canbe cloned and expanded into cell lines. This method can advantageouslybe used to engineer cell lines which express an anti-CTLA-4 antibodydescribed herein or a fragment thereof. Such engineered cell lines canbe particularly useful in screening and evaluation of compositions thatinteract directly or indirectly with the antibody molecule.

A number of selection systems can be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell11(1): 223-32), hypoxanthineguanine phosphoribosyltransferase (SzybalskaE H & Szybalski W (1962) PNAS 48(12): 2026-2034, herein incorporated byreference in its entirety) and adenine phosphoribosyltransferase (Lowy Iet al., (1980) Cell 22(3): 817-23, herein incorporated by reference inits entirety) genes in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (WiglerM et al., (1980) PNAS 77(6): 3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4): 2072-6); neo, which confers resistance tothe aminoglycoside G-418 (Wu G Y & Wu C H (1991) Biotherapy 3: 87-95;Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32: 573-596; Mulligan R C(1993) Science 260: 926-932; and Morgan R A & Anderson W F (1993) AnnRev Biochem 62: 191-217; Nabel G J & Felgner P L (1993) TrendsBiotechnol 11(5): 211-5); and hygro, which confers resistance tohygromycin (Santerre R F et al., (1984) Gene 30(1-3): 147-56), all ofwhich are herein incorporated by reference in their entireties. Methodscommonly known in the art of recombinant DNA technology can be routinelyapplied to select the desired recombinant clone and such methods aredescribed, for example, in Ausubel F M et al., (eds.), Current Protocolsin Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler M, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, N Y(1990); and in Chapters 12 and 13, Dracopoli N C et al., (eds.), CurrentProtocols in Human Genetics, John Wiley & Sons, N Y (1994);Colbère-Garapin F et al., (1981) J Mol Biol 150: 1-14, which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington C R & Hentschel C C G, Theuse of vectors based on gene amplification for the expression of clonedgenes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, NewYork, 1987), herein incorporated by reference in its entirety). When amarker in the vector system expressing antibody is amplifiable, increasein the level of inhibitor present in culture of host cell will increasethe number of copies of the marker gene. Since the amplified region isassociated with the antibody gene, production of the antibody will alsoincrease (Crouse G F et al., (1983) Mol Cell Biol 3: 257-66, hereinincorporated by reference in its entirety).

The host cell can be co-transfected with two or more expression vectorsdescribed herein, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors can contain identical selectable markerswhich enable equal expression of heavy and light chain polypeptides. Thehost cells can be co-transfected with different amounts of the two ormore expression vectors. For example, host cells can be transfected withany one of the following ratios of a first expression vector and asecond expression vector: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9,1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot N J (1986) Nature322: 562-565; and Köhler G (1980) PNAS 77: 2197-2199, which are hereinincorporated by reference in their entireties). The coding sequences forthe heavy and light chains can comprise cDNA or genomic DNA. Theexpression vector can be monocistronic or multicistronic. Amulticistronic nucleic acid construct can encode 2, 3, 4, 5, 6, 7, 8, 9,10 or more, or in the range of 2-5, 5-10 or 10-20 genes/nucleotidesequences. For example, a bicistronic nucleic acid construct cancomprise in the following order a promoter, a first gene (e.g., heavychain of an antibody described herein), and a second gene and (e.g.,light chain of an antibody described herein). In such an expressionvector, the transcription of both genes can be driven by the promoter,whereas the translation of the mRNA from the first gene can be by acap-dependent scanning mechanism and the translation of the mRNA fromthe second gene can be by a cap-independent mechanism, e.g., by an IRES.

Once an antibody molecule described herein has been produced byrecombinant expression, it can be purified by any method known in theart for purification of an immunoglobulin molecule, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of proteins. Further, theantibodies described herein can be fused to heterologous polypeptidesequences described herein or otherwise known in the art to facilitatepurification.

In specific embodiments, an antibody or an antigen-binding fragmentthereof described herein is isolated or purified. Generally, an isolatedantibody is one that is substantially free of other antibodies withdifferent antigenic specificities than the isolated antibody. Forexample, in a particular embodiment, a preparation of an antibodydescribed herein is substantially free of cellular material and/orchemical precursors. The language “substantially free of cellularmaterial” includes preparations of an antibody in which the antibody isseparated from cellular components of the cells from which it isisolated or recombinantly produced. Thus, an antibody that issubstantially free of cellular material includes preparations ofantibody having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1%(by dry weight) of heterologous protein (also referred to herein as a“contaminating protein”) and/or variants of an antibody, for example,different post-translational modified forms of an antibody or otherdifferent versions of an antibody (e.g., antibody fragments). When theantibody is recombinantly produced, it is also generally substantiallyfree of culture medium, i.e., culture medium represents less than about20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the proteinpreparation. When the antibody is produced by chemical synthesis, it isgenerally substantially free of chemical precursors or other chemicals,i.e., it is separated from chemical precursors or other chemicals whichare involved in the synthesis of the protein. Accordingly, suchpreparations of the antibody have less than about 30%, 20%, 10%, or 5%(by dry weight) of chemical precursors or compounds other than theantibody of interest. In a specific embodiment, antibodies describedherein are isolated or purified.

Antibodies or fragments thereof that specifically bind to CTLA-4 (e.g.,human CTLA-4) can be produced by any method known in the art for thesynthesis of antibodies, for example, by chemical synthesis or byrecombinant expression techniques. The methods described herein employs,unless otherwise indicated, conventional techniques in molecularbiology, microbiology, genetic analysis, recombinant DNA, organicchemistry, biochemistry, PCR, oligonucleotide synthesis andmodification, nucleic acid hybridization, and related fields within theskill of the art. These techniques are described, for example, in thereferences cited herein and are fully explained in the literature. See,e.g., Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989),Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Ausubel F M et al., Current Protocols in MolecularBiology, John Wiley & Sons (1987 and annual updates); Current Protocolsin Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.)(1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press;Eckstein (ed.) (1991) Oligonucleotides and Analogues: A PracticalApproach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: ALaboratory Manual, Cold Spring Harbor Laboratory Press, all of which areherein incorporated by reference in their entireties.

In a specific embodiment, an antibody described herein is an antibody(e.g., recombinant antibody) prepared, expressed, created or isolated byany means that involves creation, e.g., via synthesis, geneticengineering of DNA sequences. In certain embodiments, such antibodycomprises sequences (e.g., DNA sequences or amino acid sequences) thatdo not naturally exist within the antibody germline repertoire of ananimal or mammal (e.g., human) in vivo.

In one aspect, provided herein is a method of making an antibody or anantigen-binding fragment thereof which specifically binds to CTLA-4(e.g., human CTLA-4) comprising culturing a cell or host cell describedherein. In a certain aspect, provided herein is a method of making anantibody or an antigen-binding fragment thereof which specifically bindsto CTLA-4 (e.g., human CTLA-4) comprising expressing (e.g.,recombinantly expressing) the antibody or antigen-binding fragmentthereof using a cell or host cell described herein (e.g., a cell or ahost cell comprising polynucleotides encoding an antibody describedherein). In a particular embodiment, the cell is an isolated cell. In aparticular embodiment, the exogenous polynucleotides have beenintroduced into the cell. In a particular embodiment, the method furthercomprises the step of purifying the antibody or antigen-binding fragmentthereof obtained from the cell or host cell. Preferably, the method isperformed in vitro.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York, hereinincorporated by reference in its entirety).

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow E & Lane D,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed. 1988); Hammerling G J et al., in: Monoclonal Antibodies andT-Cell Hybridomas 563 681 (Elsevier, N.Y., 1981), which are hereinincorporated by reference in their entireties. The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. For example, monoclonal antibodies can be producedrecombinantly from host cells exogenously expressing an antibodydescribed herein or a fragment thereof, for example, light chain and/orheavy chain of such antibody.

In specific embodiments, a “monoclonal antibody,” as used herein, is anantibody produced by a single cell (e.g., hybridoma or host cellproducing a recombinant antibody), wherein the antibody specificallybinds to CTLA-4 (e.g., human CTLA-4) as determined, e.g., by ELISA orother antigen-binding or competitive binding assay known in the art orin the examples provided herein. In particular embodiments, a monoclonalantibody can be a chimeric antibody or a humanized antibody. In certainembodiments, a monoclonal antibody is a monovalent antibody ormultivalent (e.g., bivalent) antibody. In particular embodiments, amonoclonal antibody is a monospecific or multispecific antibody (e.g.,bispecific antibody). Monoclonal antibodies described herein can, forexample, be made by the hybridoma method as described in Kohler G &Milstein C (1975) Nature 256: 495, herein incorporated by reference inits entirety, or can, e.g., be isolated from phage libraries using thetechniques as described herein, for example. Other methods for thepreparation of clonal cell lines and of monoclonal antibodies expressedthereby are well known in the art (see, for example, Chapter 11 in:Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel F M etal., supra).

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. For example,in the hybridoma method, a mouse or other appropriate host animal, suchas a sheep, goat, rabbit, rat, hamster or macaque monkey, is immunizedto elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein (e.g., CTLA-4(e.g., human CTLA-4)) used for immunization. Alternatively, lymphocytesmay be immunized in vitro. Lymphocytes then are fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (Goding J W (Ed), Monoclonal Antibodies: Principles andPractice, pp. 59-103 (Academic Press, 1986), herein incorporated byreference in its entirety). Additionally, a RIMMS (repetitiveimmunization multiple sites) technique can be used to immunize an animal(Kilpatrick K E et al., (1997) Hybridoma 16:381-9, herein incorporatedby reference in its entirety).

In some embodiments, mice (or other animals, such as rats, monkeys,donkeys, pigs, sheep, hamster, or dogs) can be immunized with an antigen(e.g., CTLA-4 (e.g., human CTLA-4)) and once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well-known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theAmerican Type Culture Collection (ATCC®) (Manassas, Va.), to formhybridomas. Hybridomas are selected and cloned by limited dilution. Incertain embodiments, lymph nodes of the immunized mice are harvested andfused with NS0 myeloma cells.

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium that preferably contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells.

Specific embodiments employ myeloma cells that fuse efficiently, supportstable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Among these myeloma cell lines are murine myeloma lines, such asNS0 cell line or those derived from MOPC-21 and MPC-11 mouse tumorsavailable from the Salk Institute Cell Distribution Center, San Diego,Calif., USA, and SP-2 or X63-Ag8.653 cells available from the AmericanType Culture Collection, Rockville, Md., USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor D (1984) J Immunol133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniquesand Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), whichare herein incorporated by reference in their entireties).

Culture medium in which hybridoma cells are growing is assayed forproduction of monoclonal antibodies directed against CTLA-4 (e.g., humanCTLA-4). The binding specificity of monoclonal antibodies produced byhybridoma cells is determined by methods known in the art, for example,immunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods(Goding J W (Ed), Monoclonal Antibodies: Principles and Practice,supra). Suitable culture media for this purpose include, for example,D-MEM or RPMI 1640 medium. In addition, the hybridoma cells may be grownin vivo as ascites tumors in an animal.

The monoclonal antibodies secreted by the subclones are suitablyseparated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Antibodies described herein include antibody fragments which recognizespecific CTLA-4 (e.g., human CTLA-4) and can be generated by anytechnique known to those of skill in the art. For example, Fab andF(ab′)₂ fragments described herein can be produced by proteolyticcleavage of immunoglobulin molecules, using enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab′)₂ fragments). A Fabfragment corresponds to one of the two identical arms of an antibodymolecule and contains the complete light chain paired with the VH andCH1 domains of the heavy chain. A F(ab′)₂ fragment contains the twoantigen-binding arms of an antibody molecule linked by disulfide bondsin the hinge region.

Further, the antibodies described herein or antigen-binding fragmentsthereof can also be generated using various phage display methods knownin the art. In phage display methods, functional antibody domains aredisplayed on the surface of phage particles which carry thepolynucleotide sequences encoding them. In particular, DNA sequencesencoding VH and VL domains are amplified from animal cDNA libraries(e.g., human or murine cDNA libraries of affected tissues). The DNAencoding the VH and VL domains are recombined together with a scFvlinker by PCR and cloned into a phagemid vector. The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13, and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to a particular antigen can be selected oridentified with antigen, e.g., using labeled antigen or antigen bound orcaptured to a solid surface or bead. Examples of phage display methodsthat can be used to make the antibodies described herein include thosedisclosed in Brinkman U et al., (1995) J Immunol Methods 182: 41-50;Ames R S et al., (1995) J Immunol Methods 184: 177-186; Kettleborough CA et al., (1994) Eur J Immunol 24: 952-958; Persic L et al., (1997) Gene187: 9-18; Burton D R & Barbas C F (1994) Advan Immunol 57: 191-280; PCTApplication No. PCT/GB91/001134; International Publication Nos. WO90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/1 1236, WO95/15982, WO 95/20401, and WO 97/13844; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and5,969,108, all of which are herein incorporated by reference in theirentireties.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceantibody fragments such as Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication No. WO 92/22324; Mullinax R L et al., (1992) BioTechniques12(6): 864-9; Sawai H et al., (1995) Am J Reprod Immunol 34: 26-34; andBetter M et al., (1988) Science 240: 1041-1043, all of which are hereinincorporated by reference in their entireties.

In certain embodiments, to generate whole antibodies, PCR primersincluding VH or VL nucleotide sequences, a restriction site, and aflanking sequence to protect the restriction site can be used to amplifythe VH or VL sequences from a template, e.g., scFv clones. Utilizingcloning techniques known to those of skill in the art, the PCR amplifiedVH domains can be cloned into vectors expressing a VH constant region,and the PCR amplified VL domains can be cloned into vectors expressing aVL constant region, e.g., human kappa or lambda constant regions. The VHand VL domains can also be cloned into one vector expressing thenecessary constant regions. The heavy chain conversion vectors and lightchain conversion vectors are then co-transfected into cell lines togenerate stable or transient cell lines that express full-lengthantibodies, e.g., IgG, using techniques known to those of skill in theart.

A chimeric antibody is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules. Forexample, a chimeric antibody can contain a variable region of a mouse orrat monoclonal antibody fused to a constant region of a human antibody.Methods for producing chimeric antibodies are known in the art. See,e.g., Morrison S L (1985) Science 229: 1202-7; Oi V T & Morrison S L(1986) BioTechniques 4: 214-221; Gillies S D et al., (1989) J ImmunolMethods 125: 191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567,4,816,397, and 6,331,415, all of which are herein incorporated byreference in their entireties.

A humanized antibody is capable of binding to a predetermined antigenand which comprises a framework region having substantially the aminoacid sequence of a human immunoglobulin and CDRs having substantiallythe amino acid sequence of a non-human immunoglobulin (e.g., a murineimmunoglobulin). In particular embodiments, a humanized antibody alsocomprises at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin. The antibody also can includethe CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. Ahumanized antibody can be selected from any class of immunoglobulins,including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgG₁,IgG₂, IgG₃ and IgG₄. Humanized antibodies can be produced using avariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592106 andEP 519596; Padlan E A (1991) Mol Immunol 28(4/5): 489-498; Studnicka G Met al., (1994) Prot Engineering 7(6): 805-814; and Roguska M A et al.,(1994) PNAS 91: 969-973), chain shuffling (U.S. Pat. No. 5,565,332), andtechniques disclosed in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No.5,766,886, International Publication No. WO 93/17105; Tan P et al.,(2002) J Immunol 169: 1119-25; Caldas C et al., (2000) Protein Eng.13(5): 353-60; Morea V et al., (2000) Methods 20(3): 267-79; Baca M etal., (1997) J Biol Chem 272(16): 10678-84; Roguska M A et al., (1996)Protein Eng 9(10): 895 904; Couto J R et al., (1995) Cancer Res. 55 (23Supp): 5973s-5977s; Couto J R et al., (1995) Cancer Res 55(8): 1717-22;Sandhu J S (1994) Gene 150(2): 409-10 and Pedersen J T et al., (1994) JMol Biol 235(3): 959-73, all of which are herein incorporated byreference in their entireties. See also U.S. Application Publication No.US 2005/0042664 A1 (Feb. 24, 2005), which is incorporated by referenceherein in its entirety.

Methods for making multispecific (e.g., bispecific antibodies) have beendescribed, see, for example, U.S. Pat. Nos. 7,951,917; 7,183,076;8,227,577; 5,837,242; 5,989,830; 5,869,620; 6,132,992 and 8,586,713, allof which are herein incorporated by reference in their entireties.

Single domain antibodies, for example, antibodies lacking the lightchains, can be produced by methods well known in the art. See RiechmannL & Muyldermans S (1999) J Immunol 231: 25-38; Nuttall S D et al.,(2000) Curr Pharm Biotechnol 1(3): 253-263; Muyldermans S, (2001) JBiotechnol 74(4): 277-302; U.S. Pat. No. 6,005,079; and InternationalPublication Nos. WO 94/04678, WO 94/25591 and WO 01/44301, all of whichare herein incorporated by reference in their entireties.

Further, antibodies that specifically bind to a CTLA-4 antigen can, inturn, be utilized to generate anti-idiotype antibodies that “mimic” anantigen using techniques well known to those skilled in the art. (See,e.g., Greenspan N S & Bona C A (1989) FASEB J 7(5): 437-444; andNissinoff A (1991) J Immunol 147(8): 2429-2438, which are hereinincorporated by reference in their entireties).

In particular embodiments, an antibody described herein, which binds tothe same epitope of CTLA-4 (e.g., human CTLA-4) as an anti-CTLA-4antibody described herein, is a human antibody or an antigen-bindingfragment thereof. In particular embodiments, an antibody describedherein, which competitively blocks (e.g., in a dose-dependent manner)any one of the antibodies described herein, from binding to CTLA-4(e.g., human CTLA-4), is a human antibody or an antigen-binding fragmentthereof. Human antibodies can be produced using any method known in theart. For example, transgenic mice which are incapable of expressingfunctional endogenous immunoglobulins, but which can express humanimmunoglobulin genes, can be used. In particular, the human heavy andlight chain immunoglobulin gene complexes can be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion can be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of theJ_(H) region prevents endogenous antibody production. The modifiedembryonic stem cells are expanded and microinjected into blastocysts toproduce chimeric mice. The chimeric mice are then bred to producehomozygous offspring which express human antibodies. The transgenic miceare immunized in the normal fashion with a selected antigen, e.g., allor a portion of an antigen (e.g., CTLA-4). Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see LonbergN & Huszar D (1995) Int Rev Immunol 13:65-93, which is hereinincorporated by reference in its entirety. For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g.,International Publication Nos. WO 98/24893, WO 96/34096 and WO 96/33735;and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318 and 5,939,598. Examples of mice capableof producing human antibodies include the Xenomouse™ (Abgenix, Inc.;U.S. Pat. Nos. 6,075,181 and 6,150,184), the HuAb-Mouse™ (Mederex,Inc./Gen Pharm; U.S. Pat. Nos. 5,545,806 and 5,569,825), the TransChromo Mouse™ (Kirin) and the KM Mouse™ (Medarex/Kirin), all of whichare herein incorporated by reference in their entireties.

Human antibodies which specifically bind to CTLA-4 (e.g., human CTLA-4)can be made by a variety of methods known in the art including phagedisplay methods described above using antibody libraries derived fromhuman immunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887,4,716,111, and 5,885,793; and International Publication Nos. WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741, all of which are herein incorporated byreference in their entireties.

In some embodiments, human antibodies can be produced using mouse-humanhybridomas. For example, human peripheral blood lymphocytes transformedwith Epstein-Barr virus (EBV) can be fused with mouse myeloma cells toproduce mouse-human hybridomas secreting human monoclonal antibodies,and these mouse-human hybridomas can be screened to determine ones whichsecrete human monoclonal antibodies that specifically bind to a targetantigen (e.g., CTLA-4 (e.g., human CTLA-4)). Such methods are known andare described in the art, see, e.g., Shinmoto H et al., (2004)Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14:27-31, which are herein incorporated by reference in their entireties.

6.6 Kits

Also provided, are kits comprising one or more antibodies describedherein, or pharmaceutical composition or conjugates thereof. In aspecific embodiment, provided herein is a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions described herein, such asone or more antibodies provided herein or an antigen-binding fragmentthereof. In some embodiments, the kits contain a pharmaceuticalcomposition described herein and any prophylactic or therapeutic agent,such as those described herein. In certain embodiments, the kits maycontain a T-cell mitogen, such as, e.g., phytohaemagglutinin (PHA)and/or phorbol myristate acetate (PMA), or a TCR complex stimulatingantibody, such as an anti-CD3 antibody and anti-CD28 antibody.Optionally associated with such container(s) can be a notice in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration.

Also provided, are kits that can be used in the above methods. In oneembodiment, a kit comprises an antibody described herein, preferably apurified antibody, in one or more containers. In a specific embodiment,kits described herein contain a substantially isolated CTLA-4 antigen(e.g., human CTLA-4) as a control. In another specific embodiment, thekits described herein further comprise a control antibody which does notreact with a CTLA-4 antigen. In another specific embodiment, kitsdescribed herein contain one or more elements for detecting the bindingof an antibody to a CTLA-4 antigen (e.g., the antibody can be conjugatedto a detectable substrate such as a fluorescent compound, an enzymaticsubstrate, a radioactive compound or a luminescent compound, or a secondantibody which recognizes the first antibody can be conjugated to adetectable substrate). In specific embodiments, a kit provided hereincan include a recombinantly produced or chemically synthesized CTLA-4antigen. The CTLA-4 antigen provided in the kit can also be attached toa solid support. In a more specific embodiment, the detecting means ofthe above described kit includes a solid support to which a CTLA-4antigen is attached. Such a kit can also include a non-attachedreporter-labeled anti-human antibody or anti-mouse/rat antibody. In thisembodiment, binding of the antibody to the CTLA-4 antigen can bedetected by binding of the said reporter-labeled antibody.

In one embodiment, the present invention relates to the use of a kit ofthe present invention for in vitro assaying and/or detection of humanCTLA-4 in a biological sample.

7. EXAMPLES

The examples in this Section (i.e., Section 6) are offered by way ofillustration, and not by way of limitation.

7.1 Example 1: Characterization of Novel Anti-CTLA-4 Antibodies

This example describes the characterization of antibodies that bind tohuman CTLA-4. In particular, this example describes the characterizationof antibodies that specifically bind to human CTLA-4 and inhibit thefunction of CTLA-4. The sequence information of the variable regions ofthese antibodies is provided in Table 4. All the antibodies wereexpressed as IgG₁ antibodies and analyzed in the assays described below.

7.1.1 Binding of Anti-CTLA-4 Antibodies to CTLA-4-Expressing Cells

Jurkat cells engineered to constitutively express human CTLA-4 (Promega)were used to analyze the binding of anti-CTLA-4 antibodies. Briefly, thecells were stained at 5×10⁵ cells/well using 2 μg/ml of antibody in a96-well plate for 30 minutes at 4° C. The cells were washed twice andincubated for 20 minutes at 4° C. with an anti-human IgG secondaryantibody (Thermo Scientific, Cat #31529). The cells were washed andsuspended in 50 μl of 2% paraformaldehyde (Alfa Aesar, Cat #43368)prepared in PBS. Data were collected with BD FACS Canto II.

As shown in FIGS. 1A-1G, all the anti-CTLA-4 antibodies tested bound toCTLA-4-expressing cells.

7.1.2 Effect of Anti-CTLA-4 Antibody on Human PBMCs FollowingStaphylococcal Enterotoxin A (SEA) Stimulation

The functional activities of the anti-CTLA-4 antibody AGEN1884.H3 (IgG₁)on primary human PBMCs were assessed following StaphylococcalEnterotoxin A (SEA) stimulation. Briefly, cryopreserved PBMCs werestimulated with 100 ng/ml of the SEA superantigen (Toxin Technologies,Cat# at101red) in the absence or presence of 10 μg/ml of an anti-CTLA-4antibody or an isotype control antibody (IgG₁) for 5 days at 37° C. and5% CO₂. IL-2 concentrations in the culture supernatant were analyzed byAlphaLISA (Perkin Elmer, Cat# AL221F).

The anti-CTLA-4 antibody AGEN1884.H3 (IgG₁) increased IL-2 production inhuman PBMCs stimulated with the SEA superantigen (FIG. 2).

7.1.3 Effect of Anti-CTLA-4 Antibody on IL-2-Luciferase Reporter CellLine

Next, the functional activities of the anti-CTLA-4 antibody AGEN1884.H3(IgG₁) were further analyzed using an IL-2-luciferase reporter assay.Briefly, a human T cell line (Jurkat) that endogenously expressed CD3and CD28 was engineered to constitutively express cell surface CTLA-4and a luciferase reporter gene driven by an IL-2 promoter. The Jurkatreporter cell line was co-cultured with an antigen presenting cell line(Raji) that endogenously expressed CD80 and CD86 and was engineered toexpress a proprietary T cell activator (Promega). T cell receptor (TCR)triggering (signal 1) was achieved by the T cell activator; andcostimulatory signaling (signal 2) was provided in trans by CD80 andCD86 expressed on Raji cells. TCR signaling in the Jurkat T cell linetriggered IL-2 expression, leading to luciferase production, a surrogatemarker for T cell activation. Co-culture of these two cell linesresulted in engagement of the inhibitory co-receptor CTLA-4 (expressedon Jurkat cells) with its natural ligands CD80 and CD86 (expressed onRaji cells) inhibiting T cell activation, demonstrated by a lack ofluciferase expression. This inhibition was relieved upon addition ofincreasing concentrations of anti-CTLA-4 blocking antibodies. Luciferaseexpression was quantified using Bio-Glo™ reagent and the resulting datawere used to determine fold response values (fold increase withAGEN1884.H3 (IgG₁) compared with an isotype control antibody (IgG₁)).

As shown in FIG. 3, the anti-CTLA-4 antibody AGEN1884.H3 (IgG₁)dose-dependently released CTLA-4 mediated inhibition of T cells in thisIL-2-luciferase reporter assay.

7.1.4 Effect of Anti-CTLA-4 Antibody on Fc Gamma Receptor IIIA ReporterCell Line

The ability of anti-CTLA-4 antibody to co-engage CTLA-4 and signal viaactivating Fc gamma receptors was evaluated using a reporter cell lineexpressing Fc gamma receptor IIIA (FcγRIIIA) (Promega). Briefly, Jurkatcells were engineered to constitutively express human CTLA-4 on the cellsurface. These target cells were co-cultured with an effector cell line(Jurkat) engineered to express FcγRIIIA (the V158 variant) upstream ofan NFAT response element (RE) driving expression of firefly luciferase.A titrated dose of AGEN1884.H3 (IgG₁) or an isotype control antibody(IgG₁) was added to the co-culture and incubated at 37° C. overnight.Simultaneous engagement of AGEN1884.H3 by the target cell line (bindingto CTLA-4 by the Fab region) and effector cell line (binding to FcγRIIIAby the Fc region) triggers NFAT RE reporter gene activation andluciferase expression. The next day, Bio-Glo reagent (Promega) was addedto the co-culture, luminescence was measured by EnVison Multimode PlateReader (Perkin Elmer), and relative light units (RLU) were recorded tocalculate fold response values (fold increase with AGEN1884.H3 (IgG₁)compared with an isotype control antibody (IgG₁)).

When bound to target cells expressing human CTLA-4 on the cell surface,the IgG₁ antibody AGEN1884.H3 activated FcγRIIIA signaling in theeffector cells (FIG. 4).

7.2 Example 2: Characterization of Anti-CTLA-4 Antibodies with DifferentFc Regions

This example analyzes the impact of Fc/Fc receptor interaction on thefunctional activity of anti-CTLA-4 antibodies. AGEN1884.H3 was expressedas antibodies in which the IgG₁ Fc region comprises the S239D/I332E,S239D/A330L/I332E, or L235V/F243L/R292P/Y300L/P396L mutations, numberedaccording to the EU numbering system, and tested in functional assaysdescribed below. The antibody AGEN1884.H3 (IgG₁ S239D/I332E) comprises aheavy chain comprising the amino acid sequence of SEQ ID NO: 24 and alight chain comprising the amino acid sequence of SEQ ID NO: 27. Theantibody AGEN1884.H3 (IgG₁ S239D/A330L/I332E) comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 25 and a light chaincomprising the amino acid sequence of SEQ ID NO: 27. The antibodyAGEN1884.H3 (IgG₁ L235V/F243L/R292P/Y300L/P396L) comprises a heavy chaincomprising the amino acid sequence of SEQ ID NO: 26 and a light chaincomprising the amino acid sequence of SEQ ID NO: 27. For comparison,AGEN1884 was also expressed as a wild type IgG₁ antibody, an IgG₁antibody comprising S239D/I332E or S239D/A330L/I332E mutations, numberedaccording to the EU numbering system, or an afucosylated IgG₁ antibody,and tested in some functional assays.

7.2.1 Binding of Anti-CTLA-4 Antibodies to CTLA-4-Expressing Cells

The binding of anti-CTLA-4 antibodies AGEN1884.H3 (IgG₁ S239D/I332E),AGEN1884.H3 (IgG₁ S239D/A330L/I332E), and AGEN1884.H3 (IgG₁L235V/F243L/R292P/Y300L/P396L) to CTLA-4-expressing cells wascharacterized similarly as described above. Briefly, Jurkat cellsengineered to express human CTLA-4 (Promega) were stained first with 5μg/ml of an anti-CTLA-4 antibody or an isotype control antibody and thenwith an anti-human IgG secondary antibody (Thermo Scientific, Cat#31529). The cells were analyzed using BD FACS Canto II.

As shown in FIGS. 5A-5D, AGEN1884.H3 antibodies with different Fcregions all bound to cells expressing human CTLA-4.

7.2.2 Effect of Anti-CTLA-4 Antibody on Ligand Binding to Human CTLA-4

In this example, the ability of an Fc variant anti-CTLA-4 antibodyAGEN1884.H3 (IgG₁-S239D/A330E/I332E) to block binding between humanCTLA-4 and its ligands, CD80 and CD86, was tested.

Briefly, recombinant CD80-Fc and CD86-Fc proteins were conjugated to thefluorochrome Alexa Fluor 647 (Invitrogen, A20186). Jurkat cells weretransduced with trCTLA4 (truncated intracellular domain) under thecontrol of the EF1α promoter, as described in Nakaseko et al. (J ExpMed. 1999 Sep. 20; 190(6): 765-774), thus producing a cell line thatconstitutively expressed human CTLA-4 on the cell surface.CTLA-4-expressing cells were incubated with a dose titration ofanti-CTLA-4 antibody AGEN1884.H3 (IgG₁-S239D/A330E/I332E), a referenceanti-CTLA-4 antibody, or an isotype control antibody (IgG₁). The cellswere then stained with fluorescently labelled CD80-Fc or CD86-Fcprotein. Following staining, fluorescence was analyzed using theLSRFortessa flow cytometer (BD Biosciences). FACS plots were analyzedusing a combination of FACS DIVA and WEHI Weasel software. Values wereplotted using Graphpad Prism software.

As shown in FIG. 6A, AGEN1884.H3 (IgG₁-S239D/A330E/I332E) and thereference anti-CTLA-4 antibody each blocked binding between human CTLA-4and CD80 in a dose-dependent manner, whereas isotype control antibody(IgG₁) had no effect. As shown in FIG. 6B, AGEN1884.H3(IgG₁-S239D/A330E/I332E) and the reference anti-CTLA-4 antibody eachalso blocked binding between human CTLA-4 and CD86 in a dose-dependentmanner, whereas isotype control antibody (IgG₁) had no effect. Thesedata show that AGEN1884.H3 (IgG₁-S239D/A330E/I332E) functions as aligand-blocking antibody for CTLA-4.

7.2.3 Effect of Anti-CTLA-4 Antibodies on Human PBMCs FollowingStaphylococcal Enterotoxin A (SEA) Stimulation

In this example, the impact of Fc regions on the functional activity ofanti-CTLA-4 antibodies was analyzed using the SEA stimulation assaydescribed above. In brief, human PBMCs were cultured in vitro with 100ng/ml of the SEA peptide (Toxin Technologies, Cat# at101red) in theabsence or presence of anti-CTLA-4 antibodies with different Fc regionsor an isotype control antibody. After five days, concentrations of IL-2in the culture supernatant, a marker of T cell activation, were measuredusing AlphaLISA (Perkin Elmer, Cat# AL221F).

As shown in FIG. 7A, the three AGEN1884.H3 antibodies containingmutations in the IgG₁ Fc regions, all of which enhanced binding toFcγRIIIA, stimulated more IL-2 secretion than AGEN1884.H3 with a wildtype IgG₁ Fc region.

In similar studies, AGEN1884.H3 or AGEN1884 antibodies with different Fcregions were tested in the SEA stimulation assay. IntroducingS239D/I332E, S239D/A330L/I332E, or L235V/F243L/R292P/Y300L/P396Lsubstitutions in the IgG₁ Fc region significantly enhanced thefunctional activity of AGEN1884.H3 (FIG. 7B). Similarly, AGEN1884 (IgG₁S239D/I332E), AGEN1884 (IgG₁ S239D/A330L/I332E), and afucosylatedAGEN1884 (IgG₁) enhanced IL-2 production at substantially lowerconcentrations compared to AGEN1884 with a wild type IgG₁ Fc region(FIG. 7C).

7.2.4 Effect of Anti-CTLA-4 Antibodies on ZAP70 Phosphorylation

In this example, the impact of Fc regions on the functional activity ofanti-CTLA-4 antibodies in the T cell-antigen presenting cell (APC)synapse was analyzed using an assay that measures extent ofphosphorylation of the protein tyrosine kinase ZAP70, which is recruitedto the TCR following TCR engagement, where it becomes phosphorylated andfacilitates downstream signaling events.

Briefly, human PBMCs were incubated with a suboptimal concentration ofSEA peptide and 10 μg/mL of isotype control antibody (IgG₁) or theanti-CTLA-4 antibodies AGEN1884.H3 (IgG₁), AGEN1884.H3 (IgG₁S239D/A330L/I332E), or AGEN1884.H3 (IgG₁ N297A). Cells were thenincubated at 37° C. for 0 (pre) 1, 5, 10, 30, or 60 minutes. At the endof the incubation, cells were lysed with cold 1×RIPA buffer supplementedwith a phosphatase/protease inhibitor cocktail (Cell SignalingTechnologies). Following supernatant clarification, proteinconcentration was quantified using bicinchoninic acid (BCA) analysis(Pierce Biotechnology). Cell lysates (20 μg/lane) were prepared in BoltLDS sample buffer diluted and heated for 10 minutes at 70° C. beforebeing loaded onto a 4-12% Bolt Bis Tris gels (Novex). Proteins wereseparated in 1× Bolt MOPS-buffer (ThermoFisher) and then blotted onto aPVDF membrane. Following blockade with 5% bovine serum albumin (BSA, 1hour), samples were incubated with primary anti-human rabbitphospho-ZAP70 (Tyr493)/Syk (Tyr526) antibody (Cell SignalingTechnologies) in blocking buffer overnight at 4° C. Membranes wereprobed with goat anti-rabbit secondary HRP-conjugate and visualized withSignalFire ECL reagent (Cell Signaling Technology). Images were capturedusing the Chemidoc imaging system (BioRad). As a control, total ZAP70protein was evaluated following membrane stripping with Restore™ PLUSWestern Blot Stripping Buffer. Densitometric analysis of phospho-ZAP70normalized to that of total ZAP70 was performed using Image J (WayneRasband; National Institute of Mental Health, Bethesda, Md., USA) andexpressed as the fold change relative to the isotype control treatedsamples that was incubated for 1 minute.

As shown in FIGS. 8A-8B, in the isotype control antibody sample, ZAP70phosphorylation was transiently increased within ten minutes afterstimulation and rapidly diminished with no detectable levels after 15minutes. In contrast, the addition of anti-CTLA-4 antibodies AGEN1884.H3(IgG₁) or AGEN1884.H3 (IgG₁ S239D/A330L/I332E) extended detectable ZAP70activation to 30 minutes, with the most pronounced activity and relativeabundance observed with AGEN1884.H3 (IgG₁ S239D/A330L/I332E).

7.2.5 Effect of Murine Anti-CTLA-4 Antibodies on Tumor Growth andIntratumoral Regulatory T Cell Depletion in a Mouse Model

In this example, the impact of Fc regions on the antitumor andintratumoral regulatory T cell (Treg) depletion activities ofanti-CTLA-4 antibodies was analyzed using a mouse model for colon cancer(CT26 tumor-bearing mice).

Briefly, 5×10⁴ CT26 tumor cells were suspended in 100 ml PBS andinjected subcutaneously into 6-8 week old female BALB/cJ mice (JacksonLaboratories). Following engraftment to a tumor volume of 50-80 mm³,mice were treated with a single 100 μg dose of murine anti-CTLA-4antibody 9D9 (mIgG2a), an Fc-silent variant of anti-CTLA-4 antibody 9D9(mIgG2a-N297A), an Fc variant of anti-CTLA-4 antibody 9D9(mIgG2a-S239D/A330L/I332E), or an isotype control antibody (mIgG2a).Amino acid sequences for the murine antibodies tested are shown in Table7.

TABLE 7 Amino acid sequences of murine anti-CTLA-4 antibodiesDescription Sequence SEQ ID NO Murine anti-CTLA-4EAKLQESGPVLVKPGASVKMSCKASGYTFTDY 49 antibodyYMNWVKQSHGKSLEWIGVINPYNGDTSYNQKF (mIgG2a)KGKATLTVDKSSSTAYMELNSLTSEDSAVYYC heavy chainARYYGSWFAYWGQGTLVTVSSAKTTAPSVYPL APVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQ SITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD VSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTE PVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG Murine anti-CTLA-4 EAKLQESGPVLVKPGASVKMSCKASGYTFTDY50 antibody YMNWVKQSHGKSLEWIGVINPYNGDTSYNQKF (mIgG2a-KGKATLTVDKSSSTAYMELNSLTSEDSAVYYC S239D/A330L/I332E)ARYYGSWFAYWGQGTLVTVSSAKTTAPSVYPL heavy chainAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSG SLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCP APNLLGGPDVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYN STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPLPEERTISKPKGSVRAPQVYVLPPPEEEMTKKQ VTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCS VVHEGLHNHHTTKSFSRTPG Murine anti-CTLA-4EAKLQESGPVLVKPGASVKMSCKASGYTFTDY 51 antibodyYMNWVKQSHGKSLEWIGVINPYNGDTSYNQKF (mIgG2a-N297A)KGKATLTVDKSSSTAYMELNSLTSEDSAVYYC heavy chainARYYGSWFAYWGQGTLVTVSSAKTTAPSVYPL APVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQ SITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD VSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLP APIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTE PVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG Murine anti-CTLA-4DIVMTQTTLSLPVSLGDQASISCRSSQSIVHSNG 52 antibodyNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRF (mIgG2a)SGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP light chainYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGG ASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTC EATHKTSTSPIVKSFNRNEC

In a first experiment, mice treated with antibodies were then measuredbiweekly for tumor growth. As shown in FIG. 9A, an Fc variant ofanti-CTLA-4 antibody 9D9 (shown as mIgG2a-S239D/A330L/I332E) inducedcomplete regression in all CT26 tumor-bearing mice (eight out of eightmice tested). In contrast, other variants of antibody 9D9 failed toelicit the same efficacy: antibody 9D9 (mIgG2a) itself induced completeregressions in three out of nine mice tested, and the Fc-silent variantof antibody 9D9 (mIgG2a-N297A) failed to induce regression in any of thenine mice tested.

In a second experiment, CT26 tumor-bearing mice were treated asdescribed above and then sacrificed at 0, 24, 72, or 240 hourspost-treatment for collection of tumor tissue, tumor draining lymphnodes, and spleens. Collected tissues were evaluated for FoxP3⁺ Tregexpansion by flow cytometry. Single cell suspensions were obtained bymechanical dissociation followed by filtration (70 μM cell strainer). Toreduce non-specific binding, the cells were incubated with an FcγRblocking antibody (Biolegend) in FACS buffer (PBS, 2 mM EDTA, 0.5% BSA,pH 7.2) for 15 minutes at ambient temperature. Samples were then washedtwice in FACS buffer and stained for a lineage panel of CD3, CD4, CD8,and CD25, as well as a fixable live/dead marker, for 30 minutes at 4° C.For Treg delineation, samples were then washed twice, fixed,permeabilized, and then incubated with an anti-FoxP3 antibody (FJK-16s)for 30 minutes at 4° C. Samples were analyzed using the LSRFortessa flowcytometer (BD Biosciences). FACS plots were analyzed using a combinationof FACS DIVA and WEHI Weasel software. As shown in FIG. 9B, theanti-CTLA-4 antibody 9D9 (mIgG2a) and the Fc variant anti-CTLA-4antibody 9D9 (mIgG2a-S239D/A330L/I332E) each reduced the quantities ofintratumoral FoxP3+ Tregs compared to the isotype control antibody, withthe Fc variant anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E)decreasing the quantity of intratumoral FoxP3+ Tregs most significantly.The Fc-silent variant of anti-CTLA-4 antibody 9D9 (mIgG2a-N297A) did notsubstantially reduce the quantity of intratumoral FoxP3+ Tregs relativeto the isotype control antibody. None of the treatment groups showedsubstantial changes in the quantities of intratumoral CD45+ leukocytesor CD4+ non-Tregs. The Fc variant anti-CTLA-4 antibody 9D9(mIgG2a-S239D/A330L/I332E) induced the largest increase in intratumoralCD8/Treg ratio over time, followed by antibody 9D9 (mIgG2a), and then bythe Fc-silent variant antibody 9D9 (mIgG2a-N297A) and the isotypecontrol antibody (mIgG2a).

As shown in FIG. 9C, the anti-CTLA-4 antibody 9D9 (mIgG2a), the Fcvariant anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) and theFc-silent variant of anti-CTLA-4 antibody 9D9 (mIgG2a-N297A) had nosubstantial effect on the quantities of tumor draining lymph node (TDLN)FoxP3+ Tregs compared to the isotype control antibody. Similarly, asshown in FIG. 9D, the anti-CTLA-4 antibody 9D9 (mIgG2a), the Fc variantanti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E) and Fc-silentvariant of anti-CTLA-4 antibody 9D9 (mIgG2a-N297A) had no substantialeffect on the quantities of splenic FoxP3+ Tregs compared to the isotypecontrol antibody.

7.2.6 Effect of Murine Anti-CTLA-4 Antibodies in Combination with aTumor Vaccine on Tumor Growth

In this example, the effect on tumor growth of a combination of murineanti-CTLA-4 antibodies and an HPV tumor vaccine was tested in theHPV+TC-1 syngeneic tumor mouse model.

The TC-1 cell line was developed by co-transformation of primary lungepithelial cells (C57BL/6) with c-Ha-ras and HPV-16 (E6/E7) oncogenes,as described in Lin et al. (1996, Cancer Res. 56(1): 21-26). For tumorimplantation, 2×10⁵ TC-1 cells were injected subcutaneously into 6-8week old female C57BL/6 mice (Jackson Laboratories). At each of days day5, 10 and 15 post-tumor implantation, mice were administered 100 μg ofanti-CTLA-4 antibody 9D9 (mIgG2a), an Fc variant anti-CTLA-4 antibody9D9 (mIgG2a-S239D/A330L/I332E), or an isotype control antibody (mIgG2a),in combination with a dose of HPV vaccine (HPV⁺ tumor, viral antigensE6/E7) or no additional treatment. Each dose of HPV vaccine contained 30μg of HSP protein (0.4 nM) complexed with HPV pool peptide (1.2 nM) andwas supplemented with 10 μg QS-21 Stimulon® adjuvant. After treatment,mice were assessed biweekly for tumor growth and were sacrificed whentumors reached 2000 mm³ or upon ulceration.

As shown in FIG. 10, the antitumor efficacy of anti-CTLA-4 antibody 9D9(mIgG2a) and the Fc variant anti-CTLA-4 antibody 9D9(mIgG2a-S239D/A330L/I332E) each showed improvement when administered incombination with HPV tumor vaccine. This effect was greater for the Fcvariant anti-CTLA-4 antibody (mIgG2a-S239D/A330L/I332E). In particular,the Fc variant anti-CTLA-4 antibody 9D9 (mIgG2a-S239D/A330L/I332E)induced a noticeable additional decrease in TC-1 tumor growth whencombined with the HPV tumor vaccine, relative to when the antibody wasadministered as a single agent. This additional decrease in tumor growthwas greater than that observed for the combinations of antibody 9D9(mIgG2a) or the isotype control antibody (mIgG2a) with the HPV tumorvaccine.

7.2.7 Characterization of Expanded and Activated T Cell Populations

In this example, expanded and activated T cell populations werecharacterized for gene expression and CpG methylation. In brief, naturalCD4⁺ CD25⁺ FOXP3⁺ regulatory T cells or CD4⁺ CD25^(+/−) FOXP3⁻non-regulatory T cells were isolated from peripheral blood of a healthyhuman donor, expanded, and activated. The T cells were thencharacterized for expression of FOXP3 and CTLA-4 by flow cytometry, andassessed for lineage stability by examining DNA CpG methylation at CpGregions within the FOXP3 and CTLA4 loci. As known in the art,hypomethylation at these CpG sites can be used to accurately delineateeffector versus regulatory T cell lineages (Waight et al., 2015, J.Immunol. 194(3): 878-882).

PBMCs were isolated via Ficoll gradient from healthy donor buffy coats(Research Blood Components, LLC) and were then enriched for effector Tcells (Teffs) or natural regulatory T cells (Tregs) using magnetic beadisolation (MACS, Miltenyi). The enriched Teffs or Tregs were activatedwith CD3-CD28 microbeads (1:1 bead:cell ratio; Invitrogen) and withrecombinant human IL-2 for seven days in RPMI media supplemented with10% heat-inactivated FBS at 37° C. and 5% CO₂. Following stimulation,the cells were evaluated for FOXP3 and CTLA-4 expression via flowcytometry. To reduce non-specific binding, the cells were pre-incubatedwith an FcγR blocking antibody (Biolegend) in FACS buffer (PBS, 2 mMEDTA, 0.5% BSA, pH 7.2) for 15 minutes at ambient temperature. Sampleswere then washed twice in FACS buffer and stained with a lineage panelof CD3, CD4, CD8, CD25, as well as a fixable cell death marker, for 30minutes at 4° C. To assess membrane CTLA-4 expression, CTLA-4 stainingwas conducted at 37° C. For intracellular FOXP3 and CTLA-4 staining,samples were washed twice, fixed, permeabilized, and incubated with ananti-FOXP3 antibody (PCH101) and anti-CTLA-4 antibody (BNI3),respectively, for 30 minutes at 4° C. Samples were then washed twice andanalyzed using the LSRFortessa flow cytometer (BD Biosciences). FACSplots were analyzed using a combination of FACS DIVA and WEHI Weaselsoftware. For CpG methylation analysis, total DNA was isolated fromapproximately 1×10⁵ naïve CD4⁺ T cells, activated Teffs, or activatedTregs and subjected to pyrosequencing.

As shown in FIG. 11A, a high level of FOXP3 expression was detected onactivated Tregs, as well as high levels of both intracellular andmembrane CTLA-4 expression. In contrast, activated Teffs showed reducedlevels of FOXP3, intracellular CTLA-4, and membrane CTLA-4 relative toactivated Tregs. In particular, substantially less membrane CTLA-4expression was observed for activated Teffs compared to activated Tregs.FIG. 11B further shows that activated Tregs also exhibitedhypomethylated FOXP3 and CTLA4 CpG regions compared to naïve andactivated Teffs from the same donor.

7.2.8 Effect of Anti-CTLA-4 Antibodies on Antibody Dependent CellularCytotoxicity of CTLA-4-Expressing Human T Cells

In this example, the effect of anti-CTLA-4 antibody AGEN1884.H3 (IgG₁)or Fc variants thereof on antibody dependent cellular cytotoxicity(ADCC) of human CTLA-4-expressing T cells was assessed using highcontent microscopy of caspase 3/7 activation to quantify ADCC activity.

Briefly, CTLA-4-expressing target cells were co-cultured with NK-92cells expressing FcγRIIIA, following opsonization with 10 μg/ml ofanti-CTLA-4 antibody or Fc variants thereof, as described below. In afirst experiment, Jurkat cells engineered to constitutively expresscell-surface human CTLA-4 were used as target cells. CTLA-4-expressingJurkat cells were generated by transducing the Jurkat cell line withtrCTLA4 (intracellular domain removed) under the control of the EF1αpromoter, as described in Nakaseko et al. (1999, J. Exp. Med. 190(6):765-774). In a second experiment, primary human activated effector andregulatory T cells were used as target cells. CTLA-4-expressing targetcells and FcγRIIIA-158V-expressing NK-92 cells were differentiallystained using red and blue live-cell tracers (Thermo Fisher) andco-cultured at a 1:1 cell ratio (1.5×10³ cells/well in 384-well plates).Samples were treated with 10 μg/ml of AGEN1884.H3 (IgG₁), AGEN1884.H3(IgG₁ N297A), AGEN1884.H3 (IgG₁ S239D/A330L/I332E), AGEN1884.H3 (IgG₁S267E/L328F), afucosylated AGEN1884.H3 (IgG₁), or an isotype controlantibody (IgG₁). Samples were then evaluated for the induction ofapoptosis over time by live confocal imaging of caspase 3/7 substrate,which fluoresces following cleavage by activated caspase. Sample imageswere acquired every 20 minutes for six hours. Percentage ADCC activityis measured as the number of apoptotic cells relative to the total cellcount under each condition.

As shown in FIG. 12A, the Fc variant AGEN1884.H3 (IgG₁S239D/A330L/I332E) antibody, the afucosylated AGEN1884.H3 antibody, andthe AGEN1884.H3 (IgG₁) antibody each induced substantially greater ADCCactivity in Jurkat cells engineered to express cell-surface CTLA-4relative to the AGEN1884.H3 (IgG₁ N297A) variant, the AGEN1884.H3 (IgG₁S267E/L328F) variant, and isotype control antibody (IgG₁). TheAGEN1884.H3 (IgG₁ S239D/A330L/I332E) Fc variant antibody and theafucosylated AGEN1884.H3 antibody induced greater increases in ADCCactivity compared to the AGEN1884.H3 (IgG₁) antibody. As shown in FIG.12B, the AGEN1884.H3 (IgG₁ S239D/A330L/I332E) Fc variant antibodyinduced the highest levels of ADCC in both primary human activatedeffector T cells (left panel) and primary human activated regulatory Tcells (right panel), followed by afucosylated AGEN1884.H3 antibody. TheAGEN1884.H3 (IgG₁) antibody also induced slightly higher levels of ADCCcompared to controls. The remaining antibodies tested induced little tono ADCC activity in either effector or regulatory T cells. Notably, theAGEN1884.H3 (IgG₁ S239D/A330L/I332E) Fc variant antibody and theafucosylated AGEN1884.H3 antibody each induced substantially greaterADCC in regulatory T cells compared to effector T cells.

7.2.9 Effect of Anti-CTLA-4 Antibodies in Combination with an Anti-PD-1Antibody on T Cell Functionality

In this example, the effect of anti-CTLA-4 antibodies in combinationwith an anti-PD-1 antibody on primary human T cell function wasexamined.

Briefly, PBMCs were isolated via Ficoll gradient from healthy donorbuffy coats (Research Blood Components, LLC) of two human donors. Thisexperiment was performed twice on PBMCs collected from each donor, for atotal of two replicates per donor. For each replicate, isolated PBMCswere incubated for four days under stimulatory culture conditions with adosage titration of anti-CTLA-4 antibody AGEN1884.H3 (IgG₁), an Fcvariant anti-CTLA-4 antibody AGEN1884.H3 (IgG₁ S239D/A330L/I332E), or anisotype control antibody (IgG₁), in combination with a fixed dosage (5μg/ml) of a reference anti-PD-1 antagonist antibody or an isotypecontrol antibody (IgG₄). Stimulatory culture conditions were defined ascells suspended in RPMI media, supplemented with 100 ng/ml SEAsuperantigen (Sigma-Aldrich), 10% heat-inactivated FBS at 37° C., and 5%CO₂. Following incubation, cell-free supernatants were assayed for IL-2production using an AlphaLISA immunoassay (Perkin-Elmer). Data wascollected using the EnVision® Multilabel Plate Reader (Perkin-Elmer),and the concentration of IL-2 was determined using an IL-2 standardcurve. Values were interpolated and plotted using Graphpad Prismsoftware.

As shown in FIGS. 13A-13D, the anti-CTLA-4 antibody AGEN1884.H3 (IgG₁)and the Fc variant anti-CTLA-4 antibody AGEN1884.H3 (IgG₁S239D/A330L/I332E) each induced increased IL-2 production relative toisotype controls or reference anti-PD-1 antibody alone. IL-2 productionwas further enhanced when AGEN1884.H3 or AGEN1884.H3 (IgG₁S239D/A330L/I332E) was combined with reference anti-PD-1 antibody.Whether administered with isotype control antibody or in combinationwith anti-PD-1 reference antibody, the Fc variant anti-CTLA-4 antibodyAGEN1884.H3 (IgG₁ S239D/A330L/I332E) induced a greater increase in IL-2production compared to AGEN1884.H3 (IgG₁). This effect was consistent inreplicates for the first donor (FIGS. 13A and 13B) and the second donor(FIGS. 13C and 13D).

7.3 Example 3: Epitope Mapping of Anti-CTLA-4 Antibody

The interaction of the Fab fragment of AGEN1884 (AGEN1884-Fab) with theextracellular domain of human CTLA-4 was studied by hydrogen-deuteriumexchange (HDX) mass spectrometry. CTLA-4 extracellular domain alone orin combination with AGEN1884-Fab, in phosphate buffered saline solutionat pH 7.4, was diluted with a ten-fold volume of deuterium oxidelabeling buffer and incubated for varying periods of time (0, 60, 300,1800, and 7200 seconds) at room temperature. Exchange of deuterium forhydrogen was quenched by adding one volume of 4 M guanidinehydrochloride, 0.85 M TCEP (tris(2-carboxyethyl)phosphine) buffer andthe final pH was 2.5. Samples were then subjected to on-columnpepsin/protease type XIII digestion and LC-MS analysis. Mass spectrawere recorded in MS only mode. For the calculation of deuteriumincorporation, the mass spectra for a given peptide were combined acrossthe extracted ion chromatogram peak and the weighted average m/z wascalculated. The mass increase from the mass of the native peptide (0minute) to the weighted averaged mass corresponds to the level ofdeuterium incorporation. The deuterium buildup curves over exchange timefor all the peptides were plotted for further analysis and were comparedwith HDExaminer software.

Most of the CTLA-4 peptides displayed identical or similar deuteriumlevels with and without the anti-human CTLA-4 Fab present. Severalpeptide segments, however, were found to have significantly decreaseddeuterium incorporation upon Fab binding. All the residues in thisparagraph are numbered according to SEQ ID NO: 33. Two regions, residues80-82 (QVT, SEQ ID NO: 39) and residues 135-149 (YPPPYYLGIGNGTQI, SEQ IDNO: 37), experienced strong deuterium protection when human CTLA-4 wasbound to Fab. The strongest decrease in deuterium uptake was observed atresidues 140-141 (YL) which thus appeared to be a main feature of theepitope of AGEN1884 on CTLA-4. Inspection of the sequences of human andcynomolgus monkey CTLA-4, both of which AGEN1884 binds strongly (datanot shown), reveals almost complete sequence identity in the two regionsdescribed above, except for a methionine substitution for leucine atposition 141 (FIG. 14A). In contrast, AGEN1884 does not bind to anysignificant extent to either mouse or rat CTLA-4 (data not shown) whichdiffer from human CTLA-4 at residues 140-143 (YLGI, SEQ ID NO: 34) atthree out of four positions (FIG. 14A). Further selectivity data showthat AGEN1884 binds with high specificity to human and cynomolgus monkeyCTLA-4 and not to other related CD28 family members including CD28,ICOS, BTLA, and PD-1 (data not shown). Sequence comparison among theserelated proteins shows that the non-CTLA-4 proteins all differ atresidues 140-143 (YLGI, SEQ ID NO: 34) (FIG. 14B), further supportingthe importance of this epitope to the binding of AGEN1884.

The invention is not to be limited in scope by the specific embodimentsdescribed herein. Indeed, various modifications of the invention inaddition to those described will become apparent to those skilled in theart from the foregoing description and accompanying figures. Suchmodifications are intended to fall within the scope of the appendedclaims.

All references (e.g., publications or patents or patent applications)cited herein are incorporated herein by reference in their entiretiesand for all purposes to the same extent as if each individual reference(e.g., publication or patent or patent application) was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes.

Other embodiments are within the following claims.

1. An isolated antibody that specifically binds to human CTLA-4, theantibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2, and CDRH3 and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2, and CDRL3, wherein: (a) CDRH1 comprises the amino acidsequence of SYSMN (SEQ ID NO: 10); (b) CDRH2 comprises the amino acidsequence of SISSSSSYIYYAXSVKG (SEQ ID NO: 18), wherein X is E or D; (c)CDRH3 comprises the amino acid sequence of VGLXGPFDI (SEQ ID NO: 19),wherein X is F or M; (d) CDRL1 comprises the amino acid sequence ofRASQSVSRYLG (SEQ ID NO: 15); (e) CDRL2 comprises the amino acid sequenceof GASTRAT (SEQ ID NO: 16); and (f) CDRL3 comprises the amino acidsequence of QQYGSSPWT (SEQ ID NO: 17), and wherein the CDRH1, CDRH2, andCDRH3 sequences of the antibody are not SEQ ID NOs: 10, 11, and 13,respectively.
 2. The isolated antibody of claim 1, wherein CDRH2comprises the amino acid sequence of SEQ ID NO:
 12. 3. The isolatedantibody of claim 1, wherein CDRH3 comprises the amino acid sequence ofSEQ ID NO:
 14. 4. An isolated antibody that specifically binds to humanCTLA-4, the antibody comprising a heavy chain variable region comprisingcomplementarity determining regions CDRH1, CDRH2, and CDRH3 and a lightchain variable region comprising complementarity determining regionsCDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, andCDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 10, 12,14, 15, 16, and 17, respectively.
 5. The isolated antibody of claim 1,wherein the antibody comprises a heavy chain variable region comprisingthe amino acid sequence of SEQ ID NO: 20 and/or a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 9, optionallywherein the N-terminal amino acid residue of the heavy chain variableregion and/or the light chain variable region of the antibody has beenconverted to pyroglutamate.
 6. The isolated antibody of claim 1, whereinthe antibody comprises a heavy chain variable region comprising an aminoacid sequence which is at least 75%, 80%, 85%, 90%, 95%, 99%, or 100%identical to an amino acid sequence selected from the group consistingof SEQ ID NOs: 2 and 4-8, and/or a light chain variable regioncomprising an amino acid sequence which is at least 75%, 80%, 85%, 90%,95%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 9,optionally wherein the N-terminal amino acid residue of the heavy chainvariable region and/or the light chain variable region of the antibodyhas been converted to pyroglutamate. 7-13. (canceled)
 14. An isolatedantibody that specifically binds to human CTLA-4, the antibodycomprising a heavy chain variable region and a light chain variableregion, wherein the heavy chain variable region and the light chainvariable region comprise the amino acid sequences set forth in SEQ IDNOs: 2 and 9; 3 and 9; 4 and 9; 5 and 9; 6 and 9; 7 and 9; or 8 and 9,respectively, optionally wherein the N-terminal amino acid residue ofthe heavy chain variable region and/or the light chain variable regionof the antibody has been converted to pyroglutamate. 15-17. (canceled)18. An isolated antibody that specifically binds to human CTLA-4, theantibody comprising a heavy chain and a light chain comprising the aminoacid sequences of SEQ ID NOs: 23 and 27; 24 and 27; 25 and 27; or 26 and27, respectively, optionally wherein the N-terminal amino acid residueof the heavy chain and/or the light chain of the antibody has beenconverted to pyroglutamate. 19-21. (canceled)
 22. The isolated antibodyof claim 1, wherein the antibody comprises: (a) a heavy chain constantregion selected from the group consisting of human IgG₁, IgG₂, IgG₃,IgG₄, IgA₁, and IgA₂, optionally wherein the heavy chain constant regioncomprises a human IgG heavy chain constant region that is a variant of awild type human IgG heavy chain constant region, wherein the varianthuman IgG heavy chain constant region binds to FcγRIIIA with a higheraffinity than the wild type human IgG heavy chain constant region bindsto FcγRIIIA; and/or (b) a light chain constant region selected from thegroup consisting of human Igκ and IgGλ, optionally wherein the lightchain constant region comprises the amino acid sequence of SEQ ID NO:32.
 23. The isolated antibody of claim 1, wherein the antibody comprisesan IgG₁ heavy chain constant region, optionally wherein: (a) the IgG₁heavy chain constant region comprises the amino acid sequence of SEQ IDNO: 28; (b) the amino acid sequence of the IgG₁ heavy chain constantregion comprises S239D/I332E mutations, optionally wherein the IgG₁heavy chain constant region comprises the amino acid sequence of SEQ IDNO: 29; (c) the amino acid sequence of the IgG₁ heavy chain constantregion comprises S239D/A330L/I332E mutations, optionally wherein theIgG₁ heavy chain constant region comprises the amino acid sequence ofSEQ ID NO: 30; (d) the amino acid sequence of the IgG₁ heavy chainconstant region comprises L235V/F243L/R292P/Y300L/P396L mutations,optionally wherein the IgG₁ heavy chain constant region comprises theamino acid sequence of SEQ ID NO: 31; (e) the IgG₁ heavy chain constantregion is afucosylated IgG₁; and/or (f) the IgG₁ heavy chain constantregion comprises a human IgG₁ heavy chain constant region that is avariant of a wild type human IgG₁ heavy chain constant region, whereinthe variant human IgG₁ heavy chain constant region binds to FcγRIIIAwith a higher affinity than the wild type human IgG₁ heavy chainconstant region binds to FcγRIIIA, wherein the amino acid positions arenumbered according to the EU numbering system. 24-38. (canceled)
 39. Theisolated antibody of claim 1, wherein the antibody is a human antibodyand/or a bispecific antibody.
 40. (canceled)
 41. The isolated antibodyof claim 1, wherein the antibody: (a) is antagonistic to human CTLA-4;(b) deactivates, reduces, or inhibits an activity of human CTLA-4; (c)inhibits binding of human CTLA-4 to human CD80 or human CD86; and/or (d)induces IL-2 production by peripheral blood mononuclear cells (PBMCs)stimulated with staphylococcal enterotoxin A (SEA). 42-44. (canceled)45. The isolated antibody of claim 1 conjugated to a cytotoxic agent,cytostatic agent, toxin, radionuclide, or detectable label. 46.(canceled)
 47. A pharmaceutical composition comprising the antibody ofclaim 1 and a pharmaceutically acceptable carrier or excipient.
 48. Anisolated polynucleotide encoding a heavy and/or light chain of theantibody of claim
 1. 49. A vector comprising the polynucleotide of claim48.
 50. A recombinant host cell comprising the polynucleotide of claim48.
 51. A method of producing an antibody that specifically binds tohuman CTLA-4, the method comprising culturing the host cell of claim 50so that the polynucleotide is expressed and the antibody is produced.52. A method of increasing T cell activation in response to an antigenin a subject, the method comprising administering to the subject aneffective amount of the antibody of claim
 1. 53. A method of treatingcancer in a subject, the method comprising administering to the subjectan effective amount of the antibody of claim
 1. 54-100. (canceled) 101.A method of treating an infectious disease in a subject, the methodcomprising administering to the subject an effective amount of theantibody of claim 1.